T7 RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof

Abstract

The invention relates to a T7RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof, and belongs to the technical field of gene editing. A T7RNAP guided cytosine base editor is developed in bacillus subtilis, four kinds of cytidine deaminase from different sources are selected to be fused with T7RNAP, the cytidine deaminase is fused at the N end of the T7RNAP, and a fusion body with the highest mutation activity is obtained through screening; the cytosine base editor is used for evolving a global transcriptional regulatory factor CodY, in-situ evolution of genome protein is achieved, and the expression level of bovine-derived beta-lactoglobulin (beta-Lg) is improved. According to the cytosine base editor, the operable range of the cytosine base editor on a genome is widened, multi-site base (C-T) editing can be realized, and an editing window is large.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a T7 RNA polymerase-guided Bacillus subtilis cytosine base editor and applications thereof. Background Art

[0002] In recent years, in vivo continuous directed evolution technology has provided new ideas for improving the application of strains in complex phenotype construction and multi-site genetic modification. Directed evolution optimizes protein function and even creates new functions by constructing diverse gene mutants and iteratively screening them. Traditional in vitro protein directed evolution strategies, such as error-prone PCR (epPCR), DNA shuffling, and the construction of amino acid saturation mutation libraries, are limited by low transformation efficiency, long evolutionary cycles, poor adaptability of mutants after transformation into hosts, and the inability to evolve continuously. In vivo continuous evolution technology uses in vivo hypermutation to achieve continuous mutation and functional screening under physiological conditions, significantly accelerating the in situ protein evolution process.

[0003] A variety of targeted in vivo protein continuous evolution methods have been developed based on different mutation mechanisms and target regions. For example, viral-based phage-assisted continuous evolution can induce genomic mutations; orthogonal DNA polymerase-based evolution systems use error-prone DNA polymerases to specifically introduce mutations on plasmids; and CRISPR-based targeted evolution tools, such as the EvolvR system, prime editing systems, and base editors (BEs), have been widely developed. The EvolvR system fuses a CRISPR-guided nicking enzyme (nCas9) with an error-prone DNA polymerase to create nicks and introduce mismatches in the target DNA. Prime editing systems are comprised of a catalytically impaired Cas9, an engineered reverse transcriptase, and a guide RNA (pegRNA), which recognizes the target site and edits the target DNA. Base editors, by fusing a base deaminase (BD) to dCas9 or nCas9, are used to precisely introduce mutations in targeted genomic regions. Cytosine deaminase (CD) catalyzes C>T and G>A substitutions. However, the mutation windows of these systems are usually concentrated in smaller gene regions, limiting their application to large gene fragments.

[0004] Currently, the continuous evolution strategy in Bacillus subtilis is mainly based on the CRISPR base editing evolution system. These systems are limited by a narrow editing window, and multiple gRNAs or crRNAs need to be designed when modifying long gene fragments or operons. In contrast, the continuous evolution system based on orthogonal T7RNAP has become a highlight due to its efficient continuous mutation ability and wide editing window. However, since Bacillus subtilis lacks a natural T7RNAP expression regulation system, uncontrolled overexpression of T7RNAP may affect cell growth, so its expression level needs to be optimized. In addition, the screening of cytidine deaminases is also particularly critical for achieving high mutation activity. Therefore, T7RNAP-guided cytosine base editors have not yet been developed in Bacillus subtilis. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a Bacillus subtilis cytosine base editor based on T7RNAP, which overcomes the problems of existing cytosine base editors with limited operable range on the genome, low editing efficiency and complex operation.

[0006] The first object of the present invention is to provide a Bacillus subtilis gene editing fusion protein, which contains cytidine deaminase PmCDA1 and T7 RNA polymerase fused to its C-terminus; wherein the amino acid sequence of cytidine deaminase PmCDA1 is shown in SEQ ID NO.4.

[0007] Furthermore, the amino acid sequence of T7 RNA polymerase is shown in SEQ ID NO.5.

[0008] Furthermore, the cytidine deaminase PmCDA1 and T7 RNA polymerase are connected via a connecting peptide; the sequence of the connecting peptide conforms to (GGGGS)n, where n is an integer selected from 1-10.

[0009] Furthermore, the sequence of the connecting peptide is most preferably SEQ ID NO. 6. Specifically, the present invention establishes a screening library by using 14 connecting peptides and 4 cytidine deaminases, and performs survival screening by selecting culture medium (Ery R ), and found that the best effect was achieved when PmCDA1-T7RNAP was linked via the SEQ ID NO.6 sequence.

[0010] Furthermore, the Bacillus subtilis includes modified or unmodified Bacillus subtilis 168.

[0011] The present invention provides a cytosine base editing fusion protein suitable for Bacillus subtilis. This gene editing fusion protein is only active when fused to the N-terminus of T7 RNAP and is inactive when attached to the C-terminus. The present invention fuses CBE with T7 RNA polymerase (T7RNAP) to develop a T7RNAP-guided base editing evolution system. This system utilizes the specificity of T7RNAP for the T7 promoter to precisely locate the target DNA region, where CBE introduces mutations. This fusion protein can expand the mutation window when used for gene editing, with the editing window being at least 1kb.

[0012] The second object of the present invention is to provide a Bacillus subtilis cytosine base editor, which base editor includes the Bacillus subtilis gene editing fusion protein and a T7 promoter.

[0013] Furthermore, the T7 promoter is located at the N-terminus or C-terminus of the target sequence to be edited.

[0014] Furthermore, the Bacillus subtilis gene editing fusion protein is expressed episomally or integratedly in Bacillus subtilis.

[0015] The third object of the present invention is to provide the use of the Bacillus subtilis gene editing fusion protein or the Bacillus subtilis cytosine base editor in gene editing.

[0016] Furthermore, the application includes converting C:G>T:A on the Bacillus subtilis genome.

[0017] Furthermore, the application includes the following steps: introducing a recombinant plasmid containing the Bacillus subtilis gene editing fusion protein into the Bacillus subtilis to be edited, inserting a T7 promoter into the N-terminus or C-terminus of the target sequence to be edited, culturing the obtained strain, and achieving gene editing in the cells.

[0018] The fourth object of the present invention is to provide a recombinant Bacillus subtilis containing the Bacillus subtilis gene editing fusion protein or the Bacillus subtilis cytosine base editor.

[0019] A fifth object of the present invention is to provide a method for screening mutants of regulatory factors with cytosine mutations, comprising the following steps:

[0020] S1. Connect a T7 promoter to the N-terminus or C-terminus of the wild-type regulatory factor coding sequence to be edited and integrate it into the Bacillus subtilis genome;

[0021] S2, introducing the recombinant plasmid containing the Bacillus subtilis gene editing fusion protein into the Bacillus subtilis of S1;

[0022] S3, expressing the protein regulated by the regulatory factor in the Bacillus subtilis of S2, and connecting a fluorescent reporter gene to the protein encoding gene;

[0023] S4. Cultivate the Bacillus subtilis of S3 and screen the desired regulatory factor mutants based on the fluorescence intensity.

[0024] Furthermore, the regulatory factor includes a codY regulatory factor; and the protein regulated by the regulatory factor includes β-Lg protein.

[0025] Furthermore, the sequence of the β-Lg protein is shown in SEQ ID NO.8.

[0026] Furthermore, the promoter for expressing the β-Lg protein includes P 566 Promoter.

[0027] Furthermore, the P 566 The sequence of the promoter is shown in SEQ ID NO.7.

[0028] The sixth object of the present invention is to provide a codY regulatory factor mutant obtained by the above screening method. The amino acid sequence of the codY regulatory factor mutant is shown in SEQ ID NO.9.

[0029] The seventh object of the present invention is to provide a polynucleotide encoding a codY regulatory factor mutant.

[0030] The eighth object of the present invention is to provide a recombinant plasmid carrying the polynucleotide.

[0031] The ninth object of the present invention is to provide a recombinant cell (preferably a microorganism, most preferably Bacillus subtilis) containing the polynucleotide.

[0032] The tenth object of the present invention is to provide a genetically engineered Bacillus subtilis, comprising the following modifications: genome integration and expression of the codY regulatory factor mutant encoding gene, and overexpression of the β-Lg protein encoding gene.

[0033] Furthermore, the codY regulatory factor mutant encoding gene is integrated into the amyE gene site.

[0034] Furthermore, using P 566 The promoter overexpresses the β-Lg protein encoding gene.

[0035] Furthermore, the recombinant Bacillus subtilis BSF01 was used as the starting strain for transformation.

[0036] By means of the above solution, the present invention has at least the following advantages:

[0037] The present invention designed and constructed a cytosine base editor based on T7RNAP, and optimized and screened it for Bacillus subtilis, obtaining a T7 RNA polymerase-guided cytosine base editor, namely a fusion protein composed of the cytosine base editor PmCDA1 and T7RNAP, which broadened the operable range of the cytosine base editor on the genome, can realize multi-site base editing (C→T), and has a large editing window.

[0038] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0040] Figure 1 This is a T7 RNA polymerase-guided cytosine base editor system. (a) The components of the T7 RNA polymerase-guided cytosine base editor; (b) The working principle of the T7 RNA polymerase-guided cytosine base editor.

[0041] Figure 2 Mutation rate of the T7 RNA polymerase-guided cytosine base editor system.

[0042] Figure 3 Evolutionary screening of cytosine base editors for CodY. (a) Fluorescence intensity of CodY mutants; (b) β-Lg production in shake flask fermentation of CodY mutants. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0044] Materials and reagents involved in the experiment:

[0045] DNA polymerase was purchased from Takara, restriction endonucleases and T4 ligase were purchased from New England Biolabs, plasmid extraction kits were purchased from Shanghai Biotech Co., Ltd., and PCR product nucleic acid purification kits were purchased from Thermo Scientific.

[0046] Seed cultures were grown in Luria-Bertani (LB) medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. The final concentrations of kanamycin, erythromycin, and anhydrotetracycline were 50 μg / mL and 0.75 μg / mL, respectively, and the inducer was 0.5 μM.

[0047] Shake flask fermentation was carried out using Terrific Broth (TB) medium: 5 g / L glycerol, 12 g / L tryptone, 24 g / L yeast powder, 2.3 g / L KH2PO4 and 12.54 g / L K2HPO4.

[0048] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell biology, recombinant DNA technology and related fields in the field of this technology, which have been fully described in existing literature.

[0049] The sequences involved in the examples are as follows:

[0050] hAID*Δ (SEQ ID NO. 1):

[0051] MDSLLMNRREFLYQFKNVRWAKGRRETYLCYVVKRRDSATSSFLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFISWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHGRTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRThAPOBEC3A(SEQ ID NO.2):

[0052] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0053] rAPOBEC1 (SEQ ID NO. 3):

[0054] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0055] PmCDA1(SEQ ID NO.4):

[0056] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV

[0057] T7RNAP(SEQ ID NO.5):

[0058] NTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0059] Linker peptide (SEQ ID NO.6):

[0060] GGGGSGGGGSGGGGS

[0061] Promoter P566 (SEQ ID NO.7):

[0062] gaatcgtcgacaaaaaacggcctctcgaaatagagggttgacactcttttgagaatatgttatattatcagaaa ggaggtgataaaa

[0063] β-Lg (SEQ ID NO. 8):

[0064] MLIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI

[0065] Mutant CodY-V171I / D208N (SEQ ID NO. 9):

[0066] MALLQKTRIINSMLQAAAGKPVNFKEMAETLRDVIDSNIFVVSRRGKLLGYSINQQIENDRMKKMLEDRQFPEEYTKNLFNVPETSSNLDINSEYTAFPVENRDLFQAGLTTIVPIIGGGERLGTLILS RLQDQFNDDDLILAEYGATVVGMEILREKAEEIEEEARSKAIVQMAISSLSYSELEAIEHIFEELDGNEGLLVASKIANRVGITRSVIVNALRKLESAGVIESRSLGMKGTYIKVPNNKFLIELENLKSH

[0067] Example 1 Design and construction of a T7 RNA polymerase-guided cytosine base editor

[0068] The construction and screening of T7 RNA polymerase-guided cytosine base editors mainly consist of the following two basic components: (1) a fusion protein composed of cytidine deaminases hAID*Δ, hAPOBEC3A, rAPOBEC1, PmCDA1 and T7RNAP from four different sources ( Figure 1a), namely CBE-T7RNAP, which can locate to a specific region of the genome with the help of T7RNAP and convert cytosine (C) in the target region to thymine (T) under the action of cytidine deaminase; (2) Bacillus subtilis E58 strain (using wild-type B. subtilis 168 as the starting strain, and integrating the inactivated Ery_E58G to obtain the E58 strain, its genotype: B. subtilis 168trpC2 derivate,comK::P xylA -comK,amyE::P Ery -Ery R _E58G(GGA),with a T7 promoter downstream ofEry R _E58G) genome integrated with the inactivated erythromycin gene Ery R The codon for amino acid 58 was mutated from GAA to GGA, and the T7 promoter was inserted into the inactivated Ery R The C-terminus of the gene is opposite to the coding sequence. CBE-T7 RNAP recognizes and binds to the T7 promoter, performing transcription-coupled mutation. Only when cytidine deaminase induces the conversion of C:G>T:A on the template chain, mutating "GGA" to the initial codon "GAA", can the cell recover erythromycin resistance ( Figure 1 b) Four cytidine deaminases were fused to the N-terminus of T7RNAP via a linker peptide. The amino acid sequences involved in the fusion process are shown in SEQ ID NOs: 1-6.

[0069] Example 2 Screening for Highly Active T7 RNA Polymerase-Guided Cytosine Base Editors

[0070] Using anhydrotetracycline (aTC)-inducible promoter P tet The fusion protein was expressed using a plasmid containing the temperature-sensitive replicon pE194. The screening procedure was as follows: First, the edited plasmid was transformed into Bacillus subtilis E58, plated on a plate containing kanamycin, and incubated at 30°C for 12 hours. Next, three single colonies were picked and inoculated into a 96-well plate containing 200 μL of LB medium and incubated at 30°C with shaking for 12 hours. Subsequently, the culture was transferred at a 1:1000 ratio to LB medium supplemented with 0.5 μM anhydrotetracycline (aTC) and incubated at 30°C and 750 rpm for 20 hours. Finally, the culture was diluted 100-fold and plated on selective (0.75 μg / mL erythromycin) and non-selective LB plates. After 20 hours of incubation, the colonies on both plates were counted and the mutation rate was calculated. Fluctuation analysis showed that the targeted mutation rate of PmCDA1-T7RNAP reached 1.6×10 -6spb (Substitutions per base per generation, spb), the mutation rate of hAID*Δ-T7RNAP was 8.4×10 -7 The mutation rate of spb and hAPOBEC3A-T7RNAP was 7.3×10 -7 The mutation rate of spb and rAPOBEC1-T7RNAP was 7.0×10 -7 Therefore, the combination of PmCDA1-T7RNAP is the cytosine base editor with the highest mutagenic activity.

[0071] Example 3 PmCDA1-T7RNAP cytosine base editor evolves the global transcriptional regulator CodY to improve the synthesis of β-Lg

[0072] To achieve intracellular expression of β-Lg in Bacillus subtilis, pHT01 was used as the plasmid backbone and the promoter P 566 The recombinant plasmid pHT01-P was constructed to express β-Lg. 566 β-lgb was transformed into Bacillus subtilis BSF01 (described in Synergistic regulation of chassis cell growth and screening of promoters, signal peptides, and fusion protein linkers for enhanced recombinant protein expression in Bacillus subtilis), with a shake flask yield of 0.37 g / L. The amino acid sequence of bovine β-lg is shown in SEQ ID NO: 8. Shake flask fermentation conditions: TB medium, 30°C, 750 rpm, for 30 hours.

[0073] Construction of plasmid pHT01-P 566 -lgb-gfp, using GFP to characterize the expression intensity of intracellular β-Lg. 43 -codY expression frame (the editable window length of the base editor is 1137 bp (including P 43 The T7 promoter was inserted into the downstream of the coding sequence and integrated (the integration site was the same as the amyE gene site) into the plasmid carrying PmCDA1-T7RNAP and pHT01-P 566-lgb-gfp plasmid, and obtained the recombinant strain C-CodY. Evolution was carried out in LB medium supplemented with 50μg / mL kanamycin, 5μg / mL chloramphenicol, and 0.5μM aTC. After 20h of culture, the strain was transferred and evolved for another 3 rounds. Finally, 10 colonies were selected to verify the fluorescence intensity (excitation wavelength -488nm; emission wavelength -523nm), and a mutant C4 (with significantly improved fluorescence intensity) was obtained. Figure 3 a) Sequencing analysis showed that the mutation sites of CodY in C4 strain were V171I and D208N. To verify the effect of codY mutation on β-Lg synthesis, pHT01-P 566 -lgb plasmid was transformed into C4 mutant (strain genotype: BSF01 derivate, amyE::P 43 -codY (V171I,D208N) ) were fermented in shake flasks, and the β-Lg production increased to 0.51 g / L, which was 37.84% higher than that of the non-evolved strain ( Figure 3 b).

[0074] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis gene editing fusion protein, characterized in that: The gene editing fusion protein contains cytidine deaminase PmCDA1 and T7 RNA polymerase fused to its C-terminus; wherein, the amino acid sequence of cytidine deaminase PmCDA1 is shown in SEQ ID NO.

4.

2. The Bacillus subtilis gene editing fusion protein according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The amino acid sequence of T7 RNA polymerase is shown in SEQ ID NO. 5; (2) The cytidine deaminase PmCDA1 and T7 RNA polymerase are connected by a connecting peptide; the sequence of the connecting peptide conforms to (GGGGS)n, where n is an integer selected from 1 to 10; (3) The Bacillus subtilis includes modified or unmodified Bacillus subtilis 168.

3. The Bacillus subtilis gene editing fusion protein according to claim 2, characterized in that The sequence of the connecting peptide is shown in SEQ ID NO.

6.

4. A Bacillus subtilis cytosine base editor, characterized in that: The base editor comprises the Bacillus subtilis gene editing fusion protein according to any one of claims 1 to 3 and a T7 promoter.

5. The Bacillus subtilis cytosine base editor according to claim 4, characterized in that Contains at least one of the following characteristics: (1) The T7 promoter is located at the N-terminus or C-terminus of the target sequence to be edited; (2) The Bacillus subtilis gene-edited fusion protein is expressed episomally or integratedly in Bacillus subtilis.

6. Use of the Bacillus subtilis gene editing fusion protein according to any one of claims 1 to 3 or the Bacillus subtilis cytosine base editor according to claim 4 or 5 in gene editing.

7. The use according to claim 6, characterized in that Contains at least one of the following characteristics: (1) The application includes converting the C:G>T:A sequence on the Bacillus subtilis genome; (2) The application includes the following steps: introducing a recombinant plasmid containing the Bacillus subtilis gene editing fusion protein into the Bacillus subtilis to be edited, inserting a T7 promoter into the N-terminus or C-terminus of the target sequence to be edited, and culturing the resulting strain to achieve gene editing.

8. A recombinant Bacillus subtilis containing the Bacillus subtilis gene editing fusion protein according to any one of claims 1 to 3 or the Bacillus subtilis cytosine base editor according to claim 4 or 5.

9. A method for screening mutants of regulatory factors with cytosine mutations, comprising the following steps: S1. Connect a T7 promoter to the N-terminus or C-terminus of the wild-type regulatory factor coding sequence to be edited and integrate it into the Bacillus subtilis genome; S2. Introducing a recombinant plasmid containing the Bacillus subtilis gene-editing fusion protein according to any one of claims 1 to 3 into the Bacillus subtilis of S1; S3, expressing the protein regulated by the regulatory factor in the Bacillus subtilis of S2, and connecting a fluorescent reporter gene to the protein encoding gene; S4. Cultivate the Bacillus subtilis of S3 and screen the desired regulatory factor mutants based on the fluorescence intensity.

10. The screening method according to claim 9, characterized in that Contains at least one of the following characteristics: (1) The regulatory factor includes a codY regulatory factor; the protein regulated by the regulatory factor includes a β-Lg protein; (2) The promoter expressing the β-Lg protein includes P 566 promoter; the P 566 The sequence of the promoter is shown in SEQ ID NO.

7.

11. A codY regulatory factor mutant, characterized in that: The amino acid sequence of the codY regulatory factor mutant is shown in SEQ ID NO.

9.

12. A polynucleotide encoding the codY regulatory factor mutant according to claim 11.

13. A recombinant plasmid carrying the polynucleotide according to claim 12.

14. A recombinant cell comprising the polynucleotide of claim 12.

15. A genetically engineered Bacillus subtilis, characterized in that: The method comprises the following modifications: integrating the gene encoding the codY regulatory factor mutant according to claim 11 into the genome and overexpressing the gene encoding the β-Lg protein.

16. The genetically engineered Bacillus subtilis according to claim 15, characterized in that Contains at least one of the following characteristics: (1) integrating the codY regulatory factor mutant encoding gene into the amyE gene locus; (2) Using P 566 Promoter overexpresses β-Lg protein encoding gene; the P 566 The sequence of the promoter is shown in SEQ ID NO.7; (3) The sequence of the β-Lg protein is shown in SEQ ID NO.8.

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