A bacillus subtilis replication coupled double deaminase mutagenesis system and application thereof

Abstract

The application provides a replication-coupled double deaminase mutation system (RCDM) suitable for Bacillus subtilis and application thereof. The system takes helicase loading factor DnaB as a core anchoring module, fuses adenine deaminase TadA8e to the N end of DnaB, fuses cytosine deaminase AID to the C end of DnaB, connects through a flexible linker, and co-expresses uracil-DNA glycosylase inhibitor Ugi, so as to realize efficient access to single-stranded DNA at replication forks, and meanwhile overcome the inhibition of endogenous Ung-mediated base excision repair on C to T mutation. The optimized RCDM system can produce nearly balanced C:G to T:A and A:T to G:C transition mutations, and the whole genome mutation frequency reaches (2.80±0.10)×10 ‑4 , which is 7.68×10 4 times the wild type spontaneous mutation rate.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and microbial breeding technology, specifically relating to a whole-genome continuous mutation system (RCDM) suitable for Bacillus subtilis and its application in directed evolution of industrial strains. Background Technology

[0002] Bacillus subtilis, a recognized safe (GRAS) Gram-positive industrial chassis microorganism, plays an irreplaceable role in the production of industrial enzymes, amino acids, antimicrobial peptides, and high-value-added chemicals due to its superior protein secretion capacity, clear genetic background, and strong environmental adaptability. However, many complex phenotypes with high industrial value (such as high secretion capacity, high tolerance, and broadened substrate spectrum) are usually regulated by multiple genes, and their optimization depends on the generation and accumulation of beneficial mutations at multiple sites throughout the genome. Because of the high fidelity of cellular DNA replication and repair mechanisms, the natural mutation rate of Bacillus subtilis is extremely low (approximately 10). -9 Up to 10 -10 The use of bases ( / ) severely limits the efficiency and speed of obtaining ideal strains through traditional adaptive laboratory evolution.

[0003] To accelerate bacterial evolution, traditional methods rely on physical mutagenesis (such as ultraviolet light and ARTP) or chemical mutagenesis (such as EMS and NTG). While these methods can generate a large number of random mutations at once, they have inherent drawbacks such as uncontrollable mutation sites, significant cell damage, difficulty in conducting multiple rounds of continuous evolution, and a large workload for positive mutation screening. In recent years, technologies such as multiplex automated genome engineering (MAGE) have been developed, enabling multi-site editing. However, these methods rely on the synthesis of large amounts of oligonucleotides and repeated electroporation operations, resulting in low efficiency and complex processes in Gram-positive bacteria such as Bacillus subtilis, making it difficult to achieve large-scale, continuous evolutionary screening.

[0004] With the development of the CRISPR-Cas system and base editing technology, site-directed mutagenesis tools based on deaminases (such as the cytosine deaminase AID / APOBEC family and adenine deaminase TadA variants) have been developed. However, most of these tools require fusion with the Cas9 protein to achieve targeting, and their editing range is limited to a narrow window specified by the sgRNA, failing to meet the need for rapid generation of genetic diversity across the entire genome. To overcome this limitation, research has begun exploring the fusion of deaminases with endogenous, naturally accessible "carrier" proteins that can access single-stranded DNA, enabling non-targeted random mutations during genome replication or transcription. For example, studies have successfully constructed genome-wide continuous evolution systems in *E. coli* and *Corynebacterium glutamicum* by fusing AID with DNA helicases (such as DnaB) or RNA polymerases. Similarly, in *Saccharomyces cerevisiae*, fusing deaminases with DNA replication-related proteins has also achieved a mutation rate increase of tens of thousands of times.

[0005] While the strategy of fusing deaminases with replication machinery has shown potential in other microorganisms, its application in Bacillus subtilis still faces systemic challenges. Most developed systems are based on heterologous host elements, often exhibiting expression and compatibility issues in Bacillus subtilis, leading to low mutation efficiency. Furthermore, existing tools mostly produce only a single type of base transition, resulting in a narrow mutation spectrum. Even attempts to construct bifunctional systems often suffer from poor protein fusion conformations, leading to severely unbalanced editing efficiency and failing to provide balanced and rich genetic diversity. More critically, the highly efficient base excision repair pathway mediated by Bacillus subtilis' endogenous uracil-DNA glycosidase (Ung) can rapidly reverse mutations catalyzed by cytosine deaminase, forming a major molecular barrier that severely limits the actual accumulation efficiency of C-to-T mutations. In addition, existing methods lack effective systematic design for achieving long-term stable and controllable mutation rates and seamless integration with continuous culture and high-throughput screening workflows, making it difficult to support truly automated continuous evolution.

[0006] In summary, although whole-genome random mutation technology has great conceptual potential, developing a tool platform specifically designed for Bacillus subtilis that can overcome its endogenous repair barrier, simultaneously and efficiently generate multiple types of point mutations, and stably operate in continuous evolutionary processes remains a gap that urgently needs to be filled in this technological field. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and develop a tool platform specifically designed for Bacillus subtilis, capable of overcoming its endogenous repair barrier, simultaneously and efficiently generating multiple types of point mutations, and stably operating in a continuous evolution process.

[0008] One of the objectives of this invention is to provide a replication-coupled diaminase mutagenesis system (i.e., the Bacillus subtilis replication-coupled diaminase mutagenesis system described in this invention, abbreviated as RCDM), which solves the problem in the prior art that the endogenous repair barrier of Bacillus subtilis leads to the masking of C to T mutations and the imbalance of the mutation spectrum, and achieves controllable, continuous and efficient whole-genome random mutagenesis.

[0009] Another object of the present invention is to provide the application of the RCDM system in the directed evolution of complex phenotypes (such as α-amylase secretion) in Bacillus subtilis.

[0010] To achieve the above objectives, this invention provides a Bacillus subtilis replication-coupled dual deaminase mutagenesis system (RCDM system), comprising a core anchoring module and a base deaminase. The Bacillus subtilis replication-coupled dual deaminase mutagenesis system expresses a DnaB-tethered dual deaminase fusion protein. The DnaB-tethered dual deaminase fusion protein uses the helicase loading factor DnaB as the core anchoring module, fusing adenine deaminase TadA8e to the N-terminus of DnaB and cytosine deaminase AID to the C-terminus of DnaB. The Bacillus subtilis replication-coupled dual deaminase mutagenesis system also co-expresses the uracil-DNA glycosidase inhibitor Ugi.

[0011] The Bacillus subtilis replication-coupled double deaminase mutagenesis system achieved continuous and balanced C:G to T:A and A:T to G:C transition mutations throughout the Bacillus subtilis genome.

[0012] Preferably, the DnaB-tethered double deaminase fusion protein and the uracil-DNA glycosidase inhibitor Ugi are expressed by the same plasmid. Preferably, the plasmid is an IPTG-inducible plasmid. Preferably, the plasmid is a pBAC0041 series plasmid. Preferably, the induction condition is 1 mM IPTG.

[0013] In any of the above-mentioned preferred embodiments, the Bacillus subtilis replication-coupled double deaminase mutagenesis system induces base mutations from C:G to T:A and from A:T to G:C.

[0014] Preferably, the amino acid sequence of cytosine deaminase AID is shown in SEQ ID NO.1.

[0015] Preferably, the amino acid sequence of the helicase loading factor DnaB is shown in SEQ ID NO.2.

[0016] Preferably, the amino acid sequence of adenine deaminase TadA8e is shown in SEQ ID NO.4.

[0017] Preferably, the amino acid sequence of the uracil-DNA glycosidase inhibitor Ugi is shown in SEQ ID NO. 5.

[0018] In any of the above-mentioned preferred embodiments, adenine deaminase TadA8e is fused to the N-terminus of DnaB and cytosine deaminase AID is fused to the C-terminus of DnaB, and the two are linked by a flexible linker, the amino acid sequence of which is shown in SEQ ID NO.3.

[0019] Preferably, the nucleotide encoding cytosine deaminase AID is the nucleotide shown in SEQ ID NO. 6.

[0020] Preferably, the encoding nucleotide of the helicase loading factor DnaB is the nucleotide shown in SEQ ID NO.7.

[0021] Preferably, the nucleotide encoding the flexible linker is the nucleotide shown in SEQ ID NO.8.

[0022] Preferably, the nucleotide encoding adenine deaminase TadA8e is the nucleotide shown in SEQ ID NO.9.

[0023] Preferably, the nucleotide encoding the uracil-DNA glycosidase inhibitor Ugi is the nucleotide shown in SEQ ID NO. 10.

[0024] This invention also provides a method for continuous genome-wide evolution of Bacillus subtilis according to the Bacillus subtilis replication-coupled double deaminase mutagenesis system described in any of the preceding claims. The method involves transforming the expression plasmid of the Bacillus subtilis replication-coupled double deaminase mutagenesis system into Bacillus subtilis, followed by iterative screening in a selection medium, and selecting OD... 600 The highest culture passage.

[0025] Preferably, any of the above involves screening for 20 to 30 rounds. More preferably, screening for 20, 25, or 30 rounds, or a range thereof.

[0026] Preferably, each round of screening takes 24 to 72 hours, with further screening taking 24, 48, 72 hours or a range thereof.

[0027] Preferably, the screening medium is M9 medium with soluble starch as the sole carbon source.

[0028] Preferably, the M9 medium, with soluble starch as the sole carbon source, contains 10 g / L of soluble starch.

[0029] Preferably, Bacillus subtilis carrying the RCDM expression plasmid is subjected to iterative screening in 96-well plates in M9 medium with soluble starch as the sole carbon source, for a total of 20 rounds, each round lasting 48 hours, to select OD... 600 The highest culture passage.

[0030] The preferred embodiment of the above is an M9 medium containing 6.78 g / L Na2HPO4, 3.00 g / L KH2PO4, 0.50 g / L NaCl, and 1.00 g / L NH4Cl; after sterilization, MgSO4 (1 mM) and CaCl2 (0.1 mM) are added, with 10 g / L soluble starch as the sole carbon source.

[0031] The method described above is used to enhance the secretion capacity of α-amylase. After evolution, the extracellular α-amylase activity is increased by about 30 times compared with the parent strain.

[0032] The present invention also provides the application of the Bacillus subtilis replication-coupled double deaminase mutagenesis system according to any one of the preceding claims or the method for continuous evolution of the whole genome of Bacillus subtilis according to any one of the preceding claims in the preparation of industrial production strains of Bacillus subtilis.

[0033] Preferably, the industrial-grade Bacillus subtilis strain is a Bacillus subtilis strain prepared using the Bacillus subtilis replication-coupled double deaminase mutagenesis system provided by this invention. Preferably, it is a strain capable of producing industrial enzyme preparations, amino acids, antimicrobial peptides, or high-value-added chemicals; preferably, it is a strain with high secretion capacity or high tolerance phenotype. Preferably, a Bacillus subtilis strain with high industrial value is obtained through the continuous evolution method of the whole genome of Bacillus subtilis provided by this invention.

[0034] Preferably, the application of the Bacillus subtilis replication-coupled double deaminase mutagenesis system or the Bacillus subtilis whole-genome continuous evolution method according to any one of the above claims in the preparation of high-yielding strains of Bacillus subtilis α-amylase.

[0035] The present invention also provides the DnaB-tethered double deaminase fusion protein described in any of the preceding claims.

[0036] The present invention also provides the nucleic acid encoding the DnaB-tethered double deaminase fusion protein described in any of the preceding claims.

[0037] The present invention also provides recombinant plasmids containing the RCDM system or recombinant Bacillus subtilis strains prepared using the RCDM system.

[0038] In this invention, a DnaB-tethered double deaminase fusion protein is constructed, in which TadA8e is fused to the N-terminus of DnaB and AID is fused to the C-terminus of DnaB, linked by a flexible linker. This achieves efficient access to single-stranded DNA at the replication fork, generating near-equilibrium C:G to T:A and A:T to G:C transition mutations. By co-expressing Ugi in the Bacillus subtilis replication-coupled double deaminase mutagenesis system, Ung-mediated base excision repair is inhibited, overcoming the inhibition of C to T mutations by endogenous Ung-mediated base excision repair. More preferably, the DnaB-tethered double deaminase fusion protein and Ugi are co-expressed on the same plasmid. Preferably, the plasmid contains the Pgrac promoter. Preferably, the Bacillus subtilis replication-coupled double deaminase mutagenesis system achieves sustained deamination mutations of single-stranded DNA at the replication fork through inducible expression (preferably, the Pgrac promoter; preferably, 1 mM IPTG). Preferably, the Bacillus subtilis replication-coupled double deaminase mutagenesis system is combined with growth-coupled screening (preferably, M9 medium with soluble starch as the sole carbon source; preferably, iterative passage in 96-well plates) for continuous evolution.

[0039] This invention provides a method for the continuous evolution of mutant strains to enhance amylase secretion expression. In a preferred embodiment of this invention, the evolution method includes the following steps:

[0040] 1. Using B. subtilis strains carrying empty vectors as the control group and strains carrying RCDM expression plasmids as the experimental group, independent sequential evolution experiments were carried out separately.

[0041] 2. Select single colonies from each group and inoculate them into LB liquid medium containing chloramphenicol to obtain seed culture;

[0042] 3. Transfer the seed culture to M9 liquid medium containing chloramphenicol (10 g / L soluble starch as the sole carbon source) and induce culture simultaneously;

[0043] 4. During each generation of continuous evolution, the OD was measured. 600 Value, select OD 600 The highest culture medium is used as the seed for the next generation (initial OD). 600 =0.1), using a 96-hole plate for iterative screening; preferably, the screening process consists of 20 rounds, with each round lasting 48 hours;

[0044] 5. Evolution was terminated when the experimental group showed a significantly improved growth capacity under starch as the sole carbon source.

[0045] 6. After termination, the cells were serially diluted and plated onto starch M9 solid plates. Single colonies with larger diameters were selected for subsequent enzyme activity tests.

[0046] Preferably, the amylase secretion level was determined using the DNS method (glucose standard curve). Using the mutant strain with amylase secretion expression capacity obtained through the continuous evolution method of this invention, in a culture system with starch as the sole carbon source, the α-amylase activity in the supernatant increased from 985 U / L in the parent strain to 29560 U / L, an increase of approximately 30-fold; OD 600 The increase from 0.314 to 2.875 confirms the high efficiency of the RCDM system in optimizing complex secretion phenotypes. Preferably, the amylase secretion level is determined using the DNS method for enzyme activity assay.

[0047] The optimized RCDM system of this invention achieves a genome-wide mutation frequency of (2.80±0.10)×102. -4 The mutation rate per base per generation is (2.52±0.33)×10 -5 The spontaneous mutation rate of the wild type is 7.68 × 10⁻⁶. 4 The extracellular α-amylase activity of Bacillus subtilis was increased by approximately 30-fold (from 985 U / L to 29560 U / L) after 20 iterations using the aforementioned Bacillus subtilis replication-coupled double deaminase mutagenesis system combined with starch growth-coupled screening. This invention provides an efficient, continuous, genome-wide non-directional mutagenesis tool for the rapid optimization of complex phenotypes in Bacillus subtilis. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the RCDM system described in this invention (TadA8e-DnaB-AID + Ugi).

[0049] Figure 2 The preferred embodiments 1 and 2 of this invention show the topology-dependent monodeaminase mutation activity and mutation spectrum of DnaB fusion.

[0050] Figure 3 In the preferred embodiment 3 of this invention, Rif is used to restore the balanced mutation profile of dideaminases by co-expressing Ugi. R Experimental results.

[0051] Figure 4 The distribution and mutation spectrum of RCDM genome-wide mutations in preferred embodiment 3 of the present invention.

[0052] Figure 5 This is a flowchart of the growth coupling continuous evolution process in the preferred embodiment 4 of the present invention.

[0053] Figure 6This is a diagram showing the results of growth-coupled enrichment using the RCDM system to enhance the extracellular α-amylase activity in Bacillus subtilis in a preferred embodiment 4 of the present invention. Detailed Implementation

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Plasmid pBAC0041 is a laboratory-preserved plasmid used for the plasmid construction described in this invention. The nucleotide sequence of plasmid pBAC0041 is shown in SEQ ID NO.11. Escherichia coli DH5α used for plasmid construction was purchased from Beijing Qingke Biotechnology Co., Ltd. Bacillus subtilis is a strain already disclosed in the prior art, described in Yu, SiliPrice, Marcus A.Wang, YuLiu,YangGuo, YanmeiNi, XiaomengRosser, Susan J.Bi, ChanghaoWang, Meng. CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in Bacillus subtilis[J]. ACSSynthetic Biology, 2020, 9(7), and is publicly available through sharing. DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from New England Biolabs. Plasmid extraction kits were purchased from AXYGEN, and PCR product nucleic acid purification kits were purchased from Thermo Scientific.

[0055] The technical solution of the present invention is as follows:

[0056] 1. Construction of DnaB-tethered double deaminase fusion protein: TadA8e is fused to the N-terminus of DnaB, and AID is fused to the C-terminus of DnaB, linked by a flexible linker;

[0057] 2. Co-expression of Ugi on the same plasmid inhibits Ung-mediated base excision repair;

[0058] 3. Inducible expression (Pgrac promoter, IPTG 1 mM) enables sustained deamination mutation of single-stranded DNA at the replication fork;

[0059] 4. Continuous evolution was carried out by combining growth-coupled screening (M9 medium with soluble starch as the sole carbon source, and iterative subculturing of 96-well plates).

[0060] The culture medium used in this invention:

[0061] LB medium (g / L): peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L.

[0062] Solid culture medium was added with 1.5% (w / v) agar and autoclaved at 121°C for 20 min.

[0063] M9 medium (g / L): Na₂HPO₄ 6.78 g / L, KH₂PO₄ 3.00 g / L, NaCl 0.50 g / L, NH₄Cl 1.00 g / L; after sterilization, MgSO₄ (1 mM) and CaCl₂ (0.1 mM) are added, with 10 g / L soluble starch as the sole carbon source. 0.5 mM IPTG is added to the medium to induce target protein expression, with a final chloramphenicol concentration of 30 μg / mL.

[0064] The primer sequences (SEQ ID NO: 12-SEQ ID NO: 43) used in the construction of the vector of the present invention are shown in Tables 1 to 3.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Table 3

[0070]

[0071] Figure 1 The diagram shown is a schematic of the RCDM system described in this invention (TadA8e-DnaB-AID + Ugi). In the diagram, TadA8e is adenosine deaminase; AID is cytidine deaminase; BER is base excision repair; Ugi is uracil DNA glycosylation inhibitor protein; and Ung is uracil DNA glycosylation enzyme. Figure 1 In the replication-coupled diaminase mutagenesis (RCDM) of *Bacillus subtilis*, TadA8e and AID are fused to the N-terminus and C-terminus of the helicase loading factor DnaB, respectively, via flexible linkers to target replication-related single-stranded DNA at the replication fork. In this optimized architecture, TadA8e deaminates adenine to inosine, leading to an A:T to G:C conversion; while AID deaminates cytidine to uracil, potentially leading to a C:G to T:A conversion. In *Bacillus subtilis*, endogenous Ung cleaves uracil and restricts the fixation of cytidine-derived mutations; this effect can be blocked by co-expression of Ugi, which protects the G:U intermediate and restores efficient C to T mutagenesis.

[0072] Figure 2 The preferred embodiments 1 and 2 of this invention show the topology-dependent monodeaminase mutation activity and mutation spectrum of DnaB fusion. Figure 2 In the study of topology-dependent mutagenesis induced by AID and TadA8e tethered to DnaB in Bacillus subtilis, AID and TadA8e were fused to DnaB in different orientations, and the results were determined by the frequency of rifampicin-resistant mutations. The results showed that DnaB-AID exhibited a higher cytidine mutagenesis rate than AID-DnaB, while adenine mutagenesis required the opposite fusion orientation and was most pronounced in TadA8e-DnaB. Sequencing of independently rifampicin-resistant (RifR) isolates revealed that the active AID fusion variant underwent only the C:G to T:A conversion, while the active TadA8e fusion variant underwent only the A:T to G:C conversion. The bottom of the figure shows representative results from the RifR colony formation assay.

[0073] Figure 3 In the preferred embodiment 3 of this invention, Rif is used to restore the balanced mutation profile of dideaminases by co-expressing Ugi. R Experimental results. Figure 3 The figure showing the results of cytidine mutagenesis in Bacillus subtilis in restoring DnaB-tethered dideaminase constructs using Ugi demonstrates how different topological arrangements of TadA8e and AID around DnaB generated dideaminase and polydeaminase fusions. The TadA8e-DnaB-AID construct retained strong mutagenic activity but only produced A:T to G:C substitutions; alternative constructs (including AID-TadA8e-DnaB and DnaB-AID-TadA8e) shifted the mutation profile towards C:G to T:A transitions, failing to restore both types of mutations simultaneously. Similarly, adding a second copy of AID failed to restore detectable C to T mutagenesis. In contrast, co-expression of Ugi with TadA8e-DnaB-AID protected against cytidine-derived damage and restored C:G to T:A and A:T to G:C transitions in near-equilibrium proportions. The bottom of the figure shows representative rifampicin resistance. R Results of the colony formation assay. These results indicate that Ung-dependent uracil excision prevents the fixation of cytidine-derived mutations in the dideminase construct, therefore achieving balanced dideminase mutagenesis in Bacillus subtilis requires inhibition of related DNA repair processes.

[0074] Figure 4This is the genome-wide mutation distribution and mutation spectrum of RCDM in preferred embodiment 3 of the present invention, where A is the genome-wide mutation distribution and B is the mutation spectrum. Figure 4 The graphs showing the whole-genome mutation output of replication-coupled dideaminase mutagenesis (RCDM) in Bacillus subtilis are as follows: Figure A shows the distribution of mutations obtained from evolutionary mutants across the whole genome, exhibiting extensive chromosomal diversity, which is consistent with the replication-coupled whole-genome mutagenesis mechanism; Figure B shows the mutation spectrum, indicating that conversion mutations dominate the output results, including C:G to T:A and A:T.

[0075] Figure 5 This is a flowchart of the growth coupling continuous evolution process in the preferred embodiment 4 of the present invention. Figure 5 The diagram illustrates a continuous enrichment process using M9 medium with soluble starch as the sole carbon source in a 96-well plate, where the fastest-growing wells are selected for the next round of inoculation at each passage.

[0076] Figure 6 The figure shows the results of growth-coupled enrichment using the RCDM system to enhance extracellular α-amylase activity in Bacillus subtilis, where A represents the growth density (OD) of the strain after 20 generations of continuous evolution. 600 A shows the trend of change; B is the standard curve for reducing sugars in the DNS method; C shows the increase in α-amylase activity. A indicates that the final OD600 value gradually increases with the increase of the number of passages in vivo, indicating that the population fitness under starch selection pressure has been improved; B is the standard curve used for reducing sugar quantification in DNS detection; C shows that extracellular α-amylase activity increases with the increase of the number of passages, indicating that variants with enhanced extracellular enzyme production have been enriched.

[0077] Example 1

[0078] Screening for the optimal fusion site of DnaB and cytosine deaminase AID (detailed construction and activity verification).

[0079] DNA helicase loading factor DnaB breaks hydrogen bonds between dsDNA fragments during replication, unwinding the DNA fragments to form transient ssDNA, which then slides along the DNA template. DnaB acts as an "anchor protein" for cytosine deaminase (AID), positioning AID around the ssDNA during replication to deaminate cytosine, resulting in a C-to-T base transition.

[0080] 1. The N-terminus of AID (SEQ ID NO.1) and DnaB (SEQ ID NO.2) are fused using a linker (SEQ ID NO.3). The specific steps are as follows:

[0081] (1) Amplification of AID, Linker, DnaB, and plasmid backbone fragments. PCR reaction system: 50 μL system containing 25 μL Q5 DNA polymerase mix, 2.5 μL each of upstream and downstream primers, 1 μL template, and 19 μL ddH2O. PCR cycling program: pre-denaturation 98℃ 2 min; denaturation 98℃ 10 s, annealing 55℃ 10 s, extension 72℃ 2 kbp / min, 30 cycles; extension 72℃ 2 min, storage 4℃. The AID and Linker sequences are both codon-optimized sequences from Bacillus subtilis and synthesized by Qingke Biotechnology. The plasmid backbone fragment is the pBAC0041 backbone fragment with the nucleotide sequence shown in SEQ ID NO.11.

[0082] (2) Use Gibson recombination technology to connect the above fragments and construct pBAC0041_AID-DnaB.

[0083] 2. Fuse AID with the C-terminus of DnaB using a linker to construct pBAC0041_DnaB-AID. The construction steps are the same as above, except that the primers are replaced with the corresponding C-terminal fusion primers.

[0084] 3. Testing the mutation rates of pBAC0041_AID-DnaB and pBAC0041_DnaB-AID: using rpoB / Rif... R A method for systematically determining genomic mutation rates was developed. Results showed that the mutation rate of the empty vector (plasmid pBAC0041, with a nucleotide sequence as shown in SEQ ID NO.11, was an empty vector) control was (3.55±0.20)×10⁻¹⁰. -8 The pBAC0041_AID-DnaB value was (3.55±0.20)×10⁻⁶. -8 (No significant difference); pBAC0041_DnaB-AID was (1.95±0.01)×10 -6 (Approximately 55-fold improvement) Sanger sequencing confirmed that the mutation profile represents a pure C:G to T:A transition. Therefore, pBAC0041_DnaB-AID was selected as a foundational tool for C to T sequential evolution.

[0085] Using rpoB / Rif R The method for systematically determining the genome mutation rate is as follows:

[0086] Mutation frequency was determined using the rifampicin resistance assay. To screen for rifampicin-resistant mutants, the induced cell suspension was adjusted to OD600 = 20, and 100 μL was plated onto LB agar plates containing 50 μg / mL rifampicin. To determine the total viable count, the same cell culture was diluted to OD600 = 6 × 10⁻⁶. -5 Spread 100 μL of the solution onto a non-selective LB agar plate. Incubate the plate at 37°C for 24–48 hours, then count the colonies. The mutation frequency is calculated using the following formula:

[0087] In the formula, N Rif N represents the number of colonies on a rifampicin-containing plate. LB The colony count is on a non-selective LB plate. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation.

[0088] Example 2

[0089] Screening for the optimal fusion site between DnaB and adenine deaminase TadA8e (detailed construction and activity validation).

[0090] Similar to the AID-mediated cytosine deamination mutation mechanism, the adenine deaminase (TadA8e) was fused with DnaB for expression, allowing TadA8e to be located in a single-stranded DNA region during DNA replication and to deaminate adenine, thereby inducing an A:T to G:C base substitution mutation.

[0091] 1. Fusion of TadA8e (SEQ ID NO.4) with the N-terminus of DnaB via a linker to construct pBAC0041_TadA8e-DnaB (steps are the same as in Example 1).

[0092] 2. The TadA8e and the C-terminus of DnaB are fused together using a linker to construct pBAC0041_DnaB-TadA8e.

[0093] 3. Mutation rate test: The empty vector control was (3.55±0.20)×10⁻⁶. -8 The value of pBAC0041_TadA8e-DnaB was (2.23±0.02)×10. -4 (Boost >6000-fold), pBAC0041_DnaB-TadA8e was close to background levels; Sanger sequencing confirmed a mutation profile of pure A:T to G:C. Therefore, pBAC0041_TadA8e-DnaB was selected as a foundational tool for A to G sequential evolution.

[0094] Example 3

[0095] Construction and Ugi optimization of a dual-function RCDM system (detailed topology filtering and repair tuning).

[0096] The two single-function tools mentioned above can only produce a single type of base transition, resulting in a narrow mutation spectrum. Building upon this, a ternary fusion tool, TadA8e-DnaB-AID (preserving its optimal topology), was further constructed, possessing both C-to-T and A-to-G dual base editing activities. The uracil-DNA glycosidase inhibitor Ugi was co-expressed on the mutation tool plasmid to inhibit the reverse repair of U-to-T by the base excision repair pathway, thereby significantly improving the C-to-T mutation efficiency and achieving a balance between the A-to-G and C-to-T mutation types. 1. Use the linker to fuse TadA8e and AID with the N-terminus and C-terminus of DnaB respectively to construct pBAC0041_TadA8e-DnaB-AID.

[0097] 2. Co-express Ugi on the same plasmid to construct pBAC0041_TadA8e-DnaB-AID(Ugi) (i.e., RCDM system).

[0098] 3. Mutation rate test: The mutation rate of TadA8e-DnaB-AID alone is (2.63±0.02)×10 -4 However, only A:T to G:C were produced; the mutation frequency remained at (2.80±0.10)×10 after co-expression of Ugi. -4 C:G to T:A accounted for 52.6%, and A:T to G:C accounted for 47.4%. Whole-genome sequencing further confirmed that the mutations were evenly distributed across the entire chromosome, with a mutation rate of (2.52±0.33)×10-1 per base per generation. -5 Transitional mutations were predominant (C:G to T:A 41.6%, A:T to G:C 37.5%). Therefore, pBAC0041_TadA8e-DnaB-AID(Ugi) was selected as a bifunctional in vivo continuous evolution tool plasmid.

[0099] Figure 3 By constructing plasmids pBAC0041_TadA8e-DnaB-AID, pBAC0041_AID-DnaB-TadA8e, and pBAC0041_DnaB-AID-TadA8e, the different mutation results produced by mutagenesis systems with different arrangements were compared. The construction methods of plasmids pBAC0041_TadA8e-DnaB-AID, pBAC0041_AID-DnaB-TadA8e, and pBAC0041_DnaB-AID-TadA8e were similar. Figure 4Example 3 shows the whole-genome mutation distribution and mutation spectrum of RCDM, i.e. the mutation results that were not screened using the M9 medium with the sole starch carbon source. Figure 4 The mapping method used is a method found in existing technologies. First, for whole-genome sequencing (WGS), the Illumina NovaSeq sequencing platform was used for whole-genome sequencing (WGS) in 150 bp paired-end reads mode with a sequencing depth greater than 100×. After sequencing, the reads were aligned to the B. subtilis 168 reference genome using BWA software. Subsequently, variant sites were identified and characterized using GATK software. Finally, the mutation map was drawn and visualized to obtain... Figure 4 The distribution and mutation spectrum of RCDM genome-wide mutations are shown.

[0100] Example 4

[0101] RCDM system was used to grow and continuously evolve α-amylase-high secretory strains (detailed screening process and result verification).

[0102] Example 4 provides a method for the continuous evolution of mutant strains to enhance amylase secretion expression, the specific implementation steps of which are as follows:

[0103] 1. Using B. subtilis strains carrying empty vectors (pBAC0041 as shown in SEQ ID NO.11 as the empty vector) as the control group, and using strains carrying RCDM plasmids (strains carrying pBAC0041_TadA8e-DnaB-AID(Ugi) plasmid) as the experimental group, three independent sequential evolution experiments were carried out.

[0104] 2. Select 3 single colonies from each group, inoculate them into LB liquid medium containing 30 μg / mL chloramphenicol, and culture at 37℃ and 200 rpm for 24 h to obtain seed culture.

[0105] 3. Transfer the seed culture at 1% (v / v) to M9 liquid medium containing 30 μg / mL chloramphenicol (10 g / L soluble starch as the sole carbon source), and add 1 mM IPTG for induction. Continue culturing at 37℃ and 200 rpm for 24 h.

[0106] 4. During each generation of continuous evolution, the OD was measured. 600 Value, select OD 600 The highest culture medium is used as the seed for the next generation (initial OD). 600=0.1), using a 96-hole plate for iterative screening, for a total of 20 rounds, each round lasting 48 hours.

[0107] 5. Evolution was terminated when the experimental group showed a significantly improved growth capacity under starch as the sole carbon source.

[0108] 6. After termination, serially dilute the bacterial solution from step 5 (preferably using a conventional 10-fold serial dilution) and spread it on starch M9 solid plates. Select single colonies with larger colony diameters for subsequent enzyme activity assays.

[0109] The significantly improved growth capacity refers to the strain that grows best under the same culture conditions (i.e., the strain with the largest OD value).

[0110] The screening criterion of this invention is to select the strain with the best growth (i.e., the one with the largest OD value) under the same culture conditions to enter the next round of mutation and screening.

[0111] The amylase secretion level was determined using the DNS method (glucose standard curve), a method already in use, and will not be elaborated upon here. The α-amylase activity in the supernatant increased from 985 U / L in the parental strain to 29560 U / L, an increase of approximately 30-fold; OD... 600 The value increased from 0.314 to 2.875, confirming the high efficiency of the RCDM system in optimizing complex secretory phenotypes.

[0112] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A Bacillus subtilis replication-coupled dual deaminase mutagenesis system, comprising a core anchoring module and a base deaminase, characterized in that, The Bacillus subtilis replication-coupled double deaminase mutagenesis system expresses a DnaB-tethered double deaminase fusion protein. This DnaB-tethered double deaminase fusion protein uses the helicase loading factor DnaB as its core anchoring module, fusing adenine deaminase TadA8e to the N-terminus of DnaB and cytosine deaminase AID to the C-terminus of DnaB. The Bacillus subtilis replication-coupled double deaminase mutagenesis system also co-expresses the uracil-DNA glycosidase inhibitor Ugi.

2. The Bacillus subtilis replication-coupled dual deaminase mutagenesis system as described in claim 1, characterized in that, Bacillus subtilis replication-coupled double deaminase mutagenesis system induces base mutations from C:G to T:A and from A:T to G:C.

3. The Bacillus subtilis replication-coupled dual deaminase mutagenesis system as described in claim 1, characterized in that, The amino acid sequence of cytosine deaminase AID is shown in SEQ ID NO.1; the amino acid sequence of helicase loading factor DnaB is shown in SEQ ID NO.2; the amino acid sequence of adenine deaminase TadA8e is shown in SEQ ID NO.4; and the amino acid sequence of uracil-DNA glycosidase inhibitor Ugi is shown in SEQ ID NO.

5.

4. The Bacillus subtilis replication-coupled dual deaminase mutagenesis system as described in claim 3, characterized in that, In the DnaB-tethered double deaminase fusion protein, adenine deaminase TadA8e is fused to the N-terminus of DnaB, and cytosine deaminase AID is fused to the C-terminus of DnaB, linked by a flexible linker. The amino acid sequence of the flexible linker is shown in SEQ ID NO.

3.

5. The Bacillus subtilis replication-coupled double deaminase mutagenesis system as described in claim 4, characterized in that, The nucleotide encoding cytosine deaminase AID is shown in SEQ ID NO. 6; the nucleotide encoding helicase loading factor DnaB is shown in SEQ ID NO. 7; the nucleotide encoding flexible linker is shown in SEQ ID NO. 8; the nucleotide encoding adenine deaminase TadA8e is shown in SEQ ID NO. 9; and the nucleotide encoding uracil-DNA glycosidase inhibitor Ugi is shown in SEQ ID NO.

10.

6. The method for continuous evolution of the entire genome of Bacillus subtilis using the replication-coupled dideaminase mutagenesis system according to claim 1, characterized in that, The plasmid expressing the Bacillus subtilis replication-coupled dideaminase mutagenesis system was transformed into Bacillus subtilis, and then iteratively screened for 20 to 30 rounds in a selection medium to select OD. 600 The highest culture passage.

7. The method as described in claim 6, characterized in that, The screening medium was M9 medium with soluble starch as the sole carbon source; each round of screening lasted 24 to 72 hours.

8. The application of the Bacillus subtilis replication-coupled double deaminase mutagenesis system according to any one of claims 1-5 or the method according to claim 6 or 7 in the preparation of industrial production strains of Bacillus subtilis.

9. The DnaB-tethered double deaminase fusion protein according to any one of claims 1-5.

10. The nucleic acid encoding the DnaB-tethered double deaminase fusion protein according to any one of claims 1-5.

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