Bacillus subtilis genome low mutation rate integration site and application

Abstract

The invention discloses a bacillus subtilis genome low-mutation-rate integration site and application, and belongs to the technical field of biology. The mutation rate of different integration sites of a bacillus subtilis genome is analyzed by utilizing a fluctuation test based on bacillus subtilis thyA and an endogenous promoter PthyA thereof, some low-mutation-rate integration sites are found, the relative mutation rate is lower than 15%, and the stability of the low-mutation-rate integration sites is verified through a passage test. In the aspect of biosynthesis, the genetic stability of the N-acetylneuraminic acid pathway gene neuB is improved, so that the production stability of the N-acetylneuraminic acid is improved, and the N-acetylneuraminic acid can still be efficiently produced after 76 generations of passage. In addition, by improving the genetic stability of the T7 RNA polymerase, the stability of a T7 expression system is improved, so that the T7 expression system is still stable after 55 generations of passage. Therefore, the low-mutation-rate site has important significance in improving the stability of the biological process and promoting the industrial application of the low-mutation-rate site.

Description

Technical Field

[0001] The invention relates to a low mutation rate integration site of a Bacillus subtilis genome and application thereof, belonging to the field of biotechnology. Background Art

[0002] Designing and constructing microbial cell factories using synthetic biology and metabolic engineering technologies is an important method to achieve green biomanufacturing. However, the low stability of biological processes limits their industrial-scale application. It is speculated that the low stability of biological processes is caused by population heterogeneity. Both phenotypic variation and genetic mutation can lead to population heterogeneity, resulting in the appearance of non-productive cells or low-producing cells in the fermentation population. These cells have lost their production capacity, have a light metabolic burden, grow fast, and are more easily enriched during the fermentation process, making the biological process unstable. Non-productive cells caused by phenotypic heterogeneity can resume production at unpredictable times, but genetic heterogeneity can lead to irreversible loss of production. Therefore, genetic heterogeneity is more harmful, and improving genetic stability helps to improve the robustness of biological processes. Bacillus subtilis is a recognized food safety strain (GRAS) and is widely used in the synthesis of a variety of high-value-added products such as nutritional chemicals and industrial enzymes. Improving its production stability is of great significance.

[0003] At present, large-scale combinatorial pathway methods, population quality control and product addiction systems have been developed to improve production stability by improving the genetic stability of engineered bacteria. Large-scale combinatorial pathway methods rely on efficient multi-fragment DNA in vitro assembly methods to construct a mutant library encoding pathways, and then screen genetically stable pathway constructions from them; population quality control and product addiction systems rely on biosensors that respond to target products or intermediates, and give production or high-yielding cell growth advantages through growth and synthesis coupling, and enrich production cells or high-yielding cell subpopulations during fermentation to improve the stability of biological processes. Large-scale combinatorial pathway methods rely on efficient multi-fragment DNA in vitro assembly methods and are only applicable to genes encoded on plasmids; in addition, the process of screening genetically stable pathway constructions is time-consuming and laborious; population quality control and product addiction systems rely on biosensors that respond to (intermediate) products; in addition, the introduction of these systems will bring metabolic burdens to the host, so the system itself is prone to failure due to mutations. These characteristics limit their universality. Therefore, it is of great significance to develop efficient and universal strategies to reduce genetic heterogeneity and improve production stability. Summary of the invention

[0004] Based on the limited universality of current large-scale combinatorial pathways, population quality control and product addiction systems, and the problem that population quality control and product addiction systems themselves are easily limited by mutations, the present invention provides low mutation rate sites in the Bacillus subtilis genome and their applications, providing an efficient and versatile method for improving production stability.

[0005] The first object of the present invention is to provide a method for stably integrating a target gene in Bacillus subtilis, comprising the following steps: integrating the target gene expression cassette into one or more of the following sites of the Bacillus subtilis host bacteria genome:

[0006] (1) The leading strand of gene yabC,

[0007] (2) The leading strand of the gene perR,

[0008] (3) The leading strand of gene yhzF,

[0009] (4) The leading strand of gene ftsY,

[0010] (5) The leading strand of the gene rnhC,

[0011] (6) the leading strand of gene ytlI,

[0012] (7) The leading strand of gene yvrD,

[0013] (8) The leading strand of the gene lytR,

[0014] (9) The leading strand of gene ywiC,

[0015] (10) The leading strand of the gene sacY,

[0016] (11) The leading strand of gene yxeD,

[0017] (12) The lagging strand of gene yabC,

[0018] (13) The lagging strand of gene ycgP,

[0019] (14) The lagging strand of gene ydgE,

[0020] (15) The lagging strand of gene yflK,

[0021] (16) The lagging strand of the gene perR,

[0022] (17) The lagging strand of gene ykfD,

[0023] (18) The lagging strand of the gene rnhC,

[0024] (19) Lagging strand of gene sacY.

[0025] The leading strand of the present invention refers to a new DNA strand synthesized by continuous 5'-3' polymerization in the same direction as the movement of the replication fork during DNA replication. The lagging strand, also known as the lagging strand, is a new DNA strand synthesized by discontinuous 5'-3' polymerization in the opposite direction to the movement of the replication fork during DNA replication. The two strands in the double helix structure of DNA are antiparallel. When the replication begins to unwind, the direction of one parent strand in the parent DNA molecule is 5'-3', and the direction of the other parent strand is 3'-5'. When the parent strand in the 3'-5' direction is used as a template, DNA polymerase replicates continuously along the 5'-3' direction, the replication speed is faster, and the replication is completed earlier, which is called the leading strand. The forward direction of the leading strand is consistent with the direction of travel of the replication fork. During DNA replication, the replication of the leading strand is continuously carried out. The lagging strand is opposite to the direction of movement of the replication fork and is synthesized by discontinuous 5'-3' polymerization.

[0026] Further, the NCBI number of gene yabC is GeneID: 937010, the NCBI number of gene perR is GeneID: 939227, the NCBI number of gene yhzF is GeneID: 8303073, the NCBI number of gene ftsY is GeneID: 936921, the NCBI number of gene rnhC is GeneID: 937436, the NCBI number of gene ytlI is GeneID: 938452, the NCBI number of gene yvrD is GeneID: 938584, and the NCBI number of gene lytR is GeneID: 9367 87, the NCBI number of gene ywiC is GeneID: 937048, the NCBI number of gene sacY is GeneID: 937335, the NCBI number of gene yxeD is GeneID: 937597, the NCBI number of gene yabC is GeneID: 937010, the NCBI number of gene ycgP is GeneID: 938337, the NCBI number of gene ydgE is GeneID: 939907, the NCBI number of gene yflK is GeneID: 936119, and the NCBI number of gene ykfD is GeneID: 939863.

[0027] Furthermore, gene integration is performed by homologous recombination.

[0028] The second object of the present invention is to provide an editing system for Bacillus subtilis gene integration, wherein the editing system is designed according to one or more of the following sites:

[0029] (1) The leading strand of gene yabC,

[0030] (2) The leading strand of the gene perR,

[0031] (3) The leading strand of gene yhzF,

[0032] (4) The leading strand of gene ftsY,

[0033] (5) The leading strand of the gene rnhC,

[0034] (6) the leading strand of gene ytlI,

[0035] (7) The leading strand of gene yvrD,

[0036] (8) The leading strand of the gene lytR,

[0037] (9) The leading strand of gene ywiC,

[0038] (10) The leading strand of the gene sacY,

[0039] (11) The leading strand of gene yxeD,

[0040] (12) The lagging strand of gene yabC,

[0041] (13) The lagging strand of gene ycgP,

[0042] (14) The lagging strand of gene ydgE,

[0043] (15) The lagging strand of gene yflK,

[0044] (16) The lagging strand of the gene perR,

[0045] (17) The lagging strand of gene ykfD,

[0046] (18) The lagging strand of the gene rnhC,

[0047] (19) Lagging strand of gene sacY.

[0048] Furthermore, the editing system includes a Cre-loxP system.

[0049] Furthermore, the editing system includes Cre recombinase and an expression frame containing a target gene, wherein the expression frame is located between two LoxP sites for genome integration.

[0050] Furthermore, the target gene includes but is not limited to the gene that needs to be overexpressed in product synthesis, which can be heterologous or endogenous. The present invention includes genes encoding key enzymes in the sialic acid synthesis pathway (such as sialic acid synthase gene), genes in the T7 expression system (such as T7 RNA polymerase gene, target gene after T7 promoter).

[0051] The third object of the present invention is to provide a nucleic acid molecule encoding the editing system.

[0052] The fourth object of the present invention is to provide a recombinant plasmid or gene expression cassette carrying the editing system.

[0053] A fifth object of the present invention is to provide a recombinant cell containing the nucleic acid molecule.

[0054] Furthermore, the host of the recombinant cell includes but is not limited to microorganisms, such as bacteria and fungi.

[0055] The sixth object of the present invention is to provide a recombinant Bacillus subtilis, in which a target gene is integrated into the genome of the Bacillus subtilis host bacteria using one or more of the following as integration sites:

[0056] (1) The leading strand of gene yabC,

[0057] (2) The leading strand of the gene perR,

[0058] (3) The leading strand of gene yhzF,

[0059] (4) The leading strand of gene ftsY,

[0060] (5) The leading strand of the gene rnhC,

[0061] (6) the leading strand of gene ytlI,

[0062] (7) The leading strand of gene yvrD,

[0063] (8) The leading strand of the gene lytR,

[0064] (9) The leading strand of gene ywiC,

[0065] (10) The leading strand of the gene sacY,

[0066] (11) The leading strand of gene yxeD,

[0067] (12) The lagging strand of gene yabC,

[0068] (13) The lagging strand of gene ycgP,

[0069] (14) The lagging strand of gene ydgE,

[0070] (15) The lagging strand of gene yflK,

[0071] (16) The lagging strand of the gene perR,

[0072] (17) The lagging strand of gene ykfD,

[0073] (18) The lagging strand of the gene rnhC,

[0074] (19) Lagging strand of gene sacY.

[0075] Furthermore, the Bacillus subtilis host bacteria include but are not limited to Bacillus subtilis 168. Furthermore, the target gene includes the sialic acid synthase gene neuB.

[0076] Furthermore, the recombinant Bacillus subtilis also includes one or more of the following modifications:

[0077] (1) Knockout of the gene gamA encoding acetylglucosamine deaminase,

[0078] (2) Knockout of nagA, the gene encoding acetylglucosamine deacetylase,

[0079] (3) Knockout of nagB, the gene encoding acetylglucosamine deaminase,

[0080] (4) Knockout of acetate kinase gene ackA,

[0081] (5) Knockout of the lactate dehydrogenase gene ldh,

[0082] (6) Knockout of ptsG, the glucose-specific EIICB component of the phosphotransferase system,

[0083] (7) Enhanced expression of the glucosamine synthase gene glmS,

[0084] (8) Enhanced expression of the glucosamine-6-phosphate acetyltransferase gene GNA1,

[0085] (9) Enhanced expression of N-acetylglucosamine-2-isomerase gene AGE.

[0086] Further, the expression frame of the sialic acid synthase gene neuB is shown in SEQ ID NO.4; the NCBI number of the acetylglucosamine deaminase encoding gene gamA is GeneID: 938425; the NCBI number of the acetylglucosamine deacetylase encoding gene nagA is GeneID: 936621; the NCBI number of the acetylglucosamine deaminase encoding gene nagB is GeneID: 936619; the NCBI number of the acetate kinase gene ackA is GeneID: 937347; the NCBI number of the lactate dehydrogenase gene ldh is GeneID: 938348; the NCBI number of the phosphotransferase system glucose-specific EIICB component ptsG is GeneID: 939255; the expression frame of the glucosamine synthase gene glmS is shown in SEQ ID NO.1; the expression frame of the glucosamine-6-phosphate acetyltransferase gene GNA1 is shown in SEQ ID NO.2; the expression frame of the N-acetylglucosamine-2-epimerase gene AGE is shown in SEQ ID NO.3.

[0087] Preferably, when the target gene is the sialic acid synthase gene neuB, the integration sites are the leading strand of gene ftsY (leading strand 143°), the leading strand of gene yocN (leading strand 179°), the lagging strand of gene ftsY (lagging strand 143°), and the lagging strand of gene yneT (lagging strand 165°).

[0088] The integrated engineered strain obtained by the present invention can still efficiently produce N-acetylneuraminic acid after 76 generations of subculture. It is reported that the strain that can still maintain a high-yield state after more than 67 generations of division can meet the needs of 200m 3 Fermentation in bioreactors (Reference: Rugbjerg, P., Myling-Petersen, N., Porse, A., Sarup-Lytzen, K., Sommer, MOA, 2018a. Diverse genetic error modes constrain large-scale bio-based production. Nature communications 9, 787.), which shows its potential in synthesis.

[0089] Furthermore, the target gene includes a T7RNAP encoding gene.

[0090] Furthermore, the Bacillus subtilis host bacteria includes Bacillus subtilis BSXC.

[0091] Preferably, when the target gene is a coding gene in the T7 expression system, the integration site is the lagging strand of gene yabC (lagging strand 4°), the leading strand of gene yhzF (leading strand 92°), the leading strand of gene ycgP (leading strand 30°), the lagging strand of gene lytR (lagging strand 313°), the lagging strand of gene yhzF (lagging strand 92°), the lagging strand of gene yqhB (lagging strand 219°), the lagging strand of gene yneT (lagging strand 165°), and the leading strand of gene perR (leading strand 81°).

[0092] The seventh object of the present invention is to provide a method for constructing the recombinant Bacillus subtilis, comprising the following steps: introducing Cre recombinase and a target gene expression frame with a restriction site loxP into a Bacillus subtilis host bacterium, so that the target gene expression frame replaces the gene at the integration site on the host bacterium genome, and obtaining the recombinant Bacillus subtilis.

[0093] The eighth object of the present invention is to provide the use of the method, editing system, nucleic acid molecule or recombinant plasmid or gene expression frame in Bacillus subtilis gene integration.

[0094] The ninth objective of the present invention is to provide the use of the recombinant cell or the recombinant Bacillus subtilis in biosynthesis.

[0095] Furthermore, the synthesized products include, but are not limited to, proteins, amino acids, organic acids and other substances that can be synthesized by Bacillus subtilis, such as sialic acid, or products that can be regulated and synthesized by the T7 expression system.

[0096] Beneficial effects of the present invention:

[0097] The present invention has discovered a series of new low mutation rate integration sites in Bacillus subtilis through screening and verification, and these sites can be used to improve the genetic stability of the integrated gene, thereby achieving stable production. Specifically, the present invention uses a novel low mutation rate integration site in Bacillus subtilis thyA and its endogenous promoter P thyA The mutation rate of different integration sites of the Bacillus subtilis genome was analyzed by fluctuation test, and some low mutation rate integration sites were found. Subsequently, by improving the genetic stability of the N-acetylneuraminic acid pathway gene neuB, the production stability of N-acetylneuraminic acid was improved, so that it can still efficiently produce N-acetylneuraminic acid after 76 generations. In addition, by using these sites, the stability of the T7 expression system was improved by improving the genetic stability of T7 RNA polymerase, so that it is still stable after 55 generations, further expanding the application range of low mutation rate sites. This provides an efficient and versatile method for improving the stability of biological processes. Therefore, low mutation rate sites are of great significance to improving the stability of biological processes and promoting their industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Schematic diagram of integrating the mutant reporter gene thyA into different sites in the genome.

[0099] Figure 2 This is the construction of the starting strain C1 and the production of NeuAc.

[0100] Figure 3 The results of NeuAc production by integrating neuB for five low mutation rate sites: lagging strand 4°, leading strand 81°, leading strand 92°, leading strand 250°, and lagging strand 117°.

[0101] Figure 4 NeuAc production of strains C1-S1 to C1-S5.

[0102] Figure 5 Shows the growth conditions of strains T7G and T7G-555.

[0103] Figure 6 To be driven by the promoter P 555 Stability analysis of controlled T7RNAP placed into sites with different mutation rates in the T7G genome. DETAILED DESCRIPTION

[0104] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0105] 1. Site details

[0106] Genomic location Gene Gene ID 4° YbC 937010 30° yc 938337 52° Yj Y 939907 72° Yj Y 936119 81° perR 939227 92° Yj Y 8303073 117° Yj Y 939863 127° YK 938799 143° ftS 936921 165° yneT 938220 179° YOc 939594 211° proI 938697 219° oeLh 938492 250° RN G 937436 257° Yj 938452 290° Yj Y 938584 313° LYt 936787 327° ywC 937048 337° sacY 937335 347° Yj Y 937597

[0107] 2. Primer List

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] 3. Mutation rate detection method

[0115] Streak the strain of interest onto LB agar plates. After overnight culture, inoculate a single colony into a 15 mL shake tube containing LB and culture for 5 hours. Then inoculate the culture into a 15 mL shake tube containing fresh SM (Vasantha, N., Freese, E., 1980. Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides. Journal of bacteriology 144, 1119–1125.) at a ratio of 2%. When the culture reaches OD600 = 0.4-0.6, dilute the culture 1×10 5 , and then aliquoted in 200 μl volumes into 96-well plates and then cultured at 37°C to OD600 = 0.4-0.6. In each biological replicate, at least 18 wells of 200 μl of each strain were cultured in parallel. Then 200 μl of the culture was plated on selective medium (SM agar plates supplemented with 5 mg / L trimethoprim) and cultured at 45°C. After 48 and 72 hours of culture, the number of trimethoprim-resistant mutants was counted and recorded. At the same time, four cultures were gradiently diluted and plated on complete medium (SM agar plates), and the number of colonies was counted and recorded. Next, the mutation rate per cell per generation was calculated using the Fluctuation Analysis Calculator (FALCOR) web tool. The thyA expression cassette of a single colony on each selective plate was amplified and sequenced to obtain the mutation spectrum. The mutation rate of a certain mutation type can be determined by multiplying the total mutation rate by the proportion of that mutation type in the mutation spectrum.

[0116] SM medium: 200ml 5x SM salts, 10ml 100x trace elements, 10ml 100x iron-citrate solution, 10ml glucose (50%), 10ml tryptophan (5mg / ml), add sodium L-glutamate (40%) to a final concentration of 1%, dilute to 1000ml with ddH2O, sterilize at 121℃ for 20min

[0117] 5x SM salt solution: 175g K2HPO4, 75g KH2PO4, 12.5g Na3-Citrate·2H2O, 2.5g MgSO4·7H2O, dilute to 2000ml with ddH2O, sterilize at 121℃ for 20min

[0118] 10ml 100x trace elements: 0.55g CaCl2 / 0.735g CaCl2·2H2O, 0.1g MnCl2·4H2O, 0.17g ZnCl2, 0.033g CuCl2·2H2O / 0.043g CuCl2·6H2O, 0.06g CoCl2·6H2O, 0.06g Na2MoO4·2H2O, ddH2O to 1000ml, filter with 0.22um sterile filter to sterilize

[0119] 100x iron-citrate solution: 0.0135g FeCl3·6H2O, 0.1g Na3-Citrate·3H2O, dilute to 100ml with ddH2O, filter and sterilize with 0.22um sterile filter

[0120] 4. Continuous passage to simulate industrial-scale fermentation

[0121] Continuous subculturing can simulate the industrial scale-up process, and by monitoring the production performance of the culture during the subculturing process, the production stability of the strain can be evaluated. To test the stability of NeuAc production, the relevant strains were inoculated on LB agar plates. After overnight culture, a single colony was inoculated into a 250mL shake flask containing 25mL NeuAc fermentation medium and cultured at 37°C and 220rpm for 23 hours. The culture was then inoculated into fresh fermentation medium with an inoculum size of 2% and cultured at 37°C and 220rpm for 23 hours. At the same time, the OD 600 To determine the cumulative number of cell divisions; part of the culture was mixed with 50% glycerol at a ratio of 1:2 and then stored in a -80°C refrigerator. After multiple subcultures and culture storage, all glycerol stocks were inoculated into a 24-well deep-well plate containing 1.5 mL of fermentation medium with an inoculation volume of 1%. After 72 hours of culture, the culture was harvested to determine NeuAc production. To test the stability of the plasmid expression system, a single colony cultured overnight on an LB agar plate was inoculated into a 250 mL shake flask containing 25 mL of LB and cultured at 37°C and 220 rpm for 11 hours. Then, subculture was performed with an inoculation volume of 2%, and subculture and OD were measured every 11 hours. 600 Record and culture storage. Finally, all glycerol stocks were inoculated into 96-well plates filled with fresh LB at 1% inoculum. After 12 h of culture, the cultures were harvested to measure GFP fluorescence and OD 600 .

[0122] 5. Materials and methods involved in the present invention:

[0123] DNA polymerase was purchased from Takara, and PCR product nucleic acid purification kit was purchased from Thermo Scientific.

[0124] Except for the analysis of N-acetylneuraminic acid production stability, all cultures were cultured in LB medium containing: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L NaCl.

[0125] The stability analysis of N-acetylneuraminic acid production used a fermentation medium containing: 12 g / L yeast powder, 6 g / L tryptone, 6 g / L (NH4)2SO4, 12.5 g / L K2HPO4·3H2O, 2.5 g / L KH2PO4, 3 g / LMgSO4·7H2O, 6 g / L urea, and 60 g / L glucose (pH 7.0).

[0126] 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 conventional techniques in related fields in the art, which have been fully described in existing literature.

[0127] Example 1 Analysis of mutation rates at different integration sites in the genome

[0128] Using Bacillus subtilis 168 as the starting strain, the original thyA copy on the genome was first knocked out. The specific implementation method is as follows: using BS168 as a template, primers ΔthyA-1.2-1f / 1r and ΔthyA-1.2-3f / 3r were used to amplify 1000bp of the upstream and downstream regions of the thyA gene as homology arms; using the tool plasmid P7Z6 (reference: Yan, X., Yu, H.-J., Hong, Q., Li, S.-P., 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Applied and environmental microbiology 74, 5556–5562.) as a template, primers were designed to amplify the bleomycin resistance gene expression cassette. Then, overlap extension PCR technology was used to connect the thyA upstream region, the resistance gene expression cassette and the thyA downstream region to obtain the thyA knockout cassette. Subsequently, the thyA knockout cassette was integrated into the Bacillus subtilis 168 genome (transformation method reference: Anagnostopoulos, C., Spizizen, J., 1961. Requirements for transformation in Bacillus subtilis. Journal of bacteriology 81, 741–746; knockout principle reference: Yan, X., Yu, H.-J., Hong, Q., Li, S.-P., 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Applied and environmental microbiology 74, 5556–5562.), and the strain ΔthyA ( Figure 1 a).

[0129] Next, we inserted the mutant reporter gene thyA into 20 different locations in the genome ( Figure 1 b), including the leading strand and the lagging strand. To prevent the inserted thyA from affecting the expression of adjacent genes, we placed the thyA expression cassette containing the resistance gene between two bidirectional terminators and then inserted it into the spacer region between the two genes in a “tail-to-tail” position ( Figure 1c) (only the 143° position is a long spacer for yneT and the downstream co-directional gene parE) To construct the thyA ectopic expression cassette, the upstream and downstream homology arms were amplified using primers specific for each site using a dilution of Bacillus subtilis 168 culture as a template. When the thyA expression cassette needs to be inserted into the leading strand of the right arm and the lagging strand of the left arm of the genome, the culture dilution of Bacillus subtilis 168 and the P7C6 plasmid (reference: Yan, X., Yu, H.-J., Hong, Q., Li, S.-P., 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Applied and environmental microbiology 74, 5556–5562.) are used as templates, and the thyA and chloramphenicol resistance genes with the original promoter and terminator are amplified using primers A-1.2-2f / r and A-1.4-3f / r, respectively, and then assembled with the upstream and downstream homologous arms of the specific site by overlap extension PCR to generate the thyA ectopic expression cassette. When it is necessary to insert the thyA expression cassette on the lagging strand of the right arm of the genome and the leading strand of the left arm of the genome, the P7C6 plasmid dilution and Bacillus subtilis 168 culture were used as templates, and the primers B-1.2-2f / r and B-1.2-3f / r were used to amplify the chloramphenicol resistance gene and thyA with the original promoter and terminator, respectively, and then assembled with the upstream and downstream homologous arms of the specific site by overlap extension PCR to generate the thyA ectopic expression cassette. Then, according to the method described by Anagnostopoulos et al. (Anagnostopoulos and Spizizen, 1961), these thyA expression cassettes were integrated into the genome of strain ΔthyA to generate strains A1~A20 and B1~B20. Next, the spontaneous mutation rate of these strains in SM was analyzed ( Figure 1 d).

[0130] In summary, we found some sites with low mutation rates, and their detailed information is shown in Table 1.

[0131] Table 1 Detailed information of low mutation rate sites

[0132]

[0133] Example 2: Using low mutation rate sites to improve the stability of N-acetylneuraminic acid production

[0134] N-acetylneuraminic acid, as one of the most common sialic acids, is widely used in medicine, food and cosmetics, and is a key precursor of sialic acid-based human milk oligosaccharides. Since 2017, N-acetylneuraminic acid has been certified as a health functional additive material in the United States, the European Union and China. Therefore, it is of great value to improve the production stability of N-acetylneuraminic acid produced by the food safety strain Bacillus subtilis. It has been reported that the introduction of the NeuAc synthesis pathway significantly affects the growth of Bacillus subtilis. Therefore, the non-producing cells caused by mutations have more growth advantages, which impairs the stability of NeuAc production and thus limits its industrialization. Therefore, we next explored the effects of sites with different mutation rates on the stability of NeuAc production.

[0135] First, we selected a NeuAc-producing strain C1 as the starting strain (reference: Zhang, X., Wang, C., Lv, X., Liu, L., Li, J., Du, G., Wang, M., Liu, Y., 2021. Engineering of synthetic multiplexed pathways for high-level N-acetylneuramin acid bioproduction. Journal of agricultural and food chemistry 69, 14868–14877). The strain was constructed by overexpressing key genes related to NeuAc synthesis (all genomic integration expression) and blocking the competing pathway and the branching pathway ( Figure 2 a). Specifically, the recombinant Bacillus subtilis C1 involves the following modifications: the N-acetylglucosamine degradation pathway genes gamA (938425), nagAB (nagA: 936621, nagB: 936619), the acetate synthesis pathway gene ackA (937347), the lactate synthesis pathway gene ldh (938348) and the phosphoenolpyruvate transport pathway gene ptsG (939255) are knocked out, and the N-acetylneuraminic acid pathway genes glmS (SEQ ID NO.1), GNA1 (SEQ ID NO.2) and AGE (SEQ ID NO.3) are strengthened in multiple copies.

[0136] Next, we simulated the industrial scale-up process by serial passages and then evaluated the stability of NeuAc production in C1 by analyzing the production performance of different cell division cultures. The results showed that the NeuAc production of each lineage of the C1 strain gradually decreased after 48 generations ( Figure 2 b). Correspondingly, the specific growth rate also gradually increases ( Figure 2 c). In C1, neuB(PxpaC The promoter-regulated neuB (sequence shown in SEQ ID NO. 4) was inserted at the 165° position of the leading strand. Figure 1 As can be seen from Figure d, the mutation rate of this site is the highest among all the analyzed sites. Previously, the selection of genomic integration sites was mainly based on yield, while ignoring the stability of production, which is why key pathway genes were inserted into high mutation rate sites, resulting in unstable production. In addition, it has been reported that the expression of NeuB enzyme in the NeuAc synthesis pathway significantly affects cell growth. Therefore, non-producing cells caused by neuB mutations have more growth advantages. These two reasons lead to the instability of NeuAc production. Therefore, we speculate that low mutation rate sites help to improve the genetic stability of neuB, thereby improving the stability of NeuAc production. Next, we selected five low mutation rate sites (lagging strand 4°, leading strand 81°, leading strand 92°, leading strand 250°, lagging strand 117°) to integrate neuB to improve the stability of NeuAc production. Unfortunately, the NeuAc titers of these five strains dropped sharply compared with C1. Figure 3 ). It has been reported that overexpression of neuB will lead to a decrease in NeuAc production (reference: Zhang, X., Wang, C., Lv, X., Liu, L., Li, J., Du, G., Wang, M., Liu, Y., 2021. Engineering of synthetic multiplexed pathways for high-level N-acetylneuraminic acid bioproduction. Journal of agricultural and food chemistry 69, 14868–14877). Due to the effect of gene dosage, the expression levels of genes at different positions in the genome are different. Therefore, we speculated that the decrease in NeuAc production was caused by changes in neuB expression. To avoid this situation, we placed neuB at the 165° position on the lagging strand and sites adjacent to the 165° position, including the 143° and 179° positions on the leading and lagging strands. As Figure 4 As shown in a, except for strain C1-S4 (whose neuB was inserted at position 179° of the lagging strand), the NeuAc production of the other five strains remained the same as that of C1. Next, the stability of NeuAc production of these five strains was analyzed by continuous passage. Figure 4As shown in b and c, C1-S1 and C1-S2, which have the lowest mutation rates at the neuB integration site, can maintain high levels of NeuAc production for 76 generations, which is 1.58 times higher than C1. C1-S3 and C1-S5, which have medium mutation rates at the neuB integration site, can maintain high levels of NeuAc production for 65 and 59 generations, which are 1.35 and 1.23 times higher than C1, respectively. The results show that low mutation rate sites can effectively improve the stability of NeuAc production.

[0137] Example 3 Stability of the T7 expression system using low mutation rate sites

[0138] The T7 expression system has been applied to the expression of recombinant proteins in a variety of hosts and even in cell-free systems due to its simple genetic composition, strong orthogonality, good controllability, and high transcriptional activity. However, the introduction of the T7 expression system often inhibits growth, making it susceptible to host mutations. This limits the application of the T7 expression system in vivo. Therefore, it is very necessary to improve the stability of the T7 expression system. To address this issue, we next explored the effect of sites with different mutation rates on the stability of the T7 expression system.

[0139] In this example, a strain of Bacillus subtilis BSXC that can be induced to a highly efficient competent state (the BSXC strain is recorded in GuoH, Tian R, WuY, et al. Facilitating stable gene integration expression and copynumber amplification in Bacillus subtilis through a reversible homologousrecombination switch. Synth Syst Biotechnol. 2024; 9(3): 577-585.) is used as the starting strain. First, we analyzed the effect of introducing the T7 expression system on cell growth. We first integrated GFP (SEQID NO.5) controlled by the T7 promoter into the 81° position of the leading strand of the BSXC genome to construct strain T7G, and then placed the T7 RNA polymerase expression frame at the 92° position of the lagging strand of the T7G genome to construct strain T7G-555. Figure 5 As can be seen in the figure, the growth of the T7G strain is almost the same as that of BSXC, while the growth of T7G-555 is significantly inhibited. Therefore, the loss-of-function mutation of T7RNAP will give the mutant a growth advantage, thereby compromising the stability of the T7 expression system. In addition, it has been reported that overexpression of T7RNAP usually kills the host or causes T7RNAP mutations. Therefore, we speculate that improving the genetic stability of the burden protein T7RNAP will help improve the stability of the T7 expression system.

[0140] To confirm the above speculation, we will next 555 The controlled T7RNAP (SEQ ID NO.6) was placed into sites with different mutation rates in the T7G genome ( Figure 6 a). We then analyzed the stability of the T7 expression system by monitoring the stability of GFP protein production during serial passages. Figure 6 As shown in b, when T7RNAP is integrated at the 4° position of the lagging strand and the 92° position of the leading strand with low mutation rates, the stability of the T7 expression system is the best, and GFP can still be efficiently expressed after 55 generations of division. Among the eight sites we selected, except for the 313° and 219° sites of the lagging strand, the lower the mutation rate, the better the stability of the T7 expression system. This shows that low mutation rate sites can effectively improve the stability of the T7 expression system. In addition, after the completion of continuous subculture, the GFP expression cassette including the T7 promoter in the subculture was amplified and sequenced. No mutations were found in the entire expression cassette, which shows that the low mutation rate sites improve the stability of the T7 expression system by enhancing the genetic stability of T7RNAP.

[0141] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for stably integrating a target gene in Bacillus subtilis, characterized in that: The following steps are involved: Integrate the target gene expression cassette into one or more of the following sites in the Bacillus subtilis host genome: (1) The leading strand of gene yabC, (2) The leading strand of the gene perR, (3) The leading strand of gene yhzF, (4) The leading strand of gene ftsY, (5) The leading strand of the gene rnhC, (6) the leading strand of gene ytlI, (7) The leading strand of gene yvrD, (8) The leading strand of the gene lytR, (9) The leading strand of gene ywiC, (10) The leading strand of the gene sacY, (11) The leading strand of gene yxeD, (12) The lagging strand of gene yabC, (13) The lagging strand of gene ycgP, (14) The lagging strand of gene ydgE, (15) The lagging strand of gene yflK, (16) The lagging strand of the gene perR, (17) The lagging strand of gene ykfD, (18) The lagging strand of the gene rnhC, (19) The lagging strand of gene sacY.

2. The method according to claim 1, characterized in that Contain at least one of the following characteristics: (i) The NCBI accession number of gene yabC is GeneID: 937010, the NCBI accession number of gene perR is GeneID: 939227, the NCBI accession number of gene yhzF is GeneID: 8303073, the NCBI accession number of gene ftsY is GeneID: 936921, the NCBI accession number of gene rnhC is GeneID: 937436, the NCBI accession number of gene ytlI is GeneID: 938452, the NCBI accession number of gene yvrD is GeneID: 938584, and the NCBI accession number of gene lytR is GeneID: 93678 7. The NCBI number of gene ywiC is GeneID: 937048, the NCBI number of gene sacY is GeneID: 937335, the NCBI number of gene yxeD is GeneID: 937597, the NCBI number of gene yabC is GeneID: 937010, the NCBI number of gene ycgP is GeneID: 938337, the NCBI number of gene ydgE is GeneID: 939907, the NCBI number of gene yflK is GeneID: 936119, and the NCBI number of gene ykfD is GeneID: 939863. ; (ii) Gene integration by homologous recombination.

3. An editing system for gene integration in Bacillus subtilis, characterized in that: The editing system is designed based on one or more of the following sites: (1) The leading strand of gene yabC, (2) The leading strand of the gene perR, (3) The leading strand of gene yhzF, (4) The leading strand of gene ftsY, (5) The leading strand of the gene rnhC, (6) the leading strand of gene ytlI, (7) The leading strand of gene yvrD, (8) The leading strand of the gene lytR, (9) The leading strand of gene ywiC, (10) The leading strand of the gene sacY, (11) The leading strand of gene yxeD, (12) The lagging strand of gene yabC, (13) The lagging strand of gene ycgP, (14) The lagging strand of gene ydgE, (15) The lagging strand of gene yflK, (16) The lagging strand of the gene perR, (17) The lagging strand of gene ykfD, (18) The lagging strand of the gene rnhC, (19) Lagging strand of gene sacY.

4. The editing system according to claim 3, characterized in that: The editing system includes the Cre-loxP system.

5. The editing system according to claim 4, characterized in that: The editing system includes Cre recombinase and an expression frame containing a target gene; the expression frame is located between two LoxP sites.

6. A nucleic acid molecule encoding the editing system according to any one of claims 3 to 5.

7. A recombinant plasmid or gene expression cassette carrying the nucleic acid molecule of claim 6.

8. A recombinant cell containing the editing system according to any one of claims 3 to 5.

9. Use of the method according to claim 1 or 2, the editing system according to any one of claims 3 to 5, the nucleic acid molecule according to claim 6, or the recombinant plasmid or gene expression cassette according to claim 7 in Bacillus subtilis gene integration.

10. A recombinant Bacillus subtilis, characterized in that: The target gene is integrated into the genome of the Bacillus subtilis host bacteria using one or more of the following as integration sites: (1) The leading strand of gene yabC, (2) The leading strand of the gene perR, (3) The leading strand of gene yhzF, (4) The leading strand of gene ftsY, (5) The leading strand of the gene rnhC, (6) the leading strand of gene ytlI, (7) The leading strand of gene yvrD, (8) The leading strand of the gene lytR, (9) The leading strand of gene ywiC, (10) The leading strand of the gene sacY, (11) The leading strand of gene yxeD, (12) The lagging strand of gene yabC, (13) The lagging strand of gene ycgP, (14) The lagging strand of gene ydgE, (15) The lagging strand of gene yflK, (16) The lagging strand of the gene perR, (17) The lagging strand of gene ykfD, (18) The lagging strand of the gene rnhC, (19) Lagging strand of gene sacY.

11. The recombinant Bacillus subtilis according to claim 10, characterized in that Contains at least one of the following characteristics: (i) the Bacillus subtilis host bacteria includes Bacillus subtilis 168 or Bacillus subtilis BSXC; (ii) the target gene includes a gene encoding a key enzyme in the product synthesis pathway or a gene in the T7 expression system; (iii) the recombinant Bacillus subtilis further comprises one or more of the following modifications: (1) Knockout of the gene gamA encoding acetylglucosamine deaminase, (2) Knockout of nagA, the gene encoding acetylglucosamine deacetylase, (3) Knockout of nagB, the gene encoding acetylglucosamine deaminase, (4) Knockout of acetate kinase gene ackA, (5) Knockout of the lactate dehydrogenase gene ldh, (6) Knockout of ptsG, the glucose-specific EIICB component of the phosphotransferase system, (7) Enhanced expression of the glucosamine synthase gene glmS, (8) Enhanced expression of the glucosamine-6-phosphate acetyltransferase gene GNA1, (9) Enhanced expression of N-acetylglucosamine-2-isomerase gene AGE.

12. The recombinant Bacillus subtilis according to claim 11, characterized in that Contains at least one of the following characteristics: (i) the target gene includes sialic acid synthase gene neuB, T7 RNAP encoding gene or the gene behind T7 promoter in T7 expression system; (ii) The expression cassette of the sialic acid synthase gene neuB is shown in SEQ ID NO.4; the NCBI number of the acetylglucosamine deaminase encoding gene gamA is GeneID: 938425; the NCBI number of the acetylglucosamine deacetylase encoding gene nagA is GeneID: 936621; the NCBI number of the acetylglucosamine deaminase encoding gene nagB is GeneID: 936619; the NCBI number of the acetate kinase gene ackA is GeneID: 937347; the NCBI number of the lactate dehydrogenase gene ldh is GeneID: 938348; the NCBI number of the phosphotransferase system glucose-specific EIICB component ptsG is GeneID: 939255; the expression cassette of the glucosamine synthase gene glmS is shown in SEQ ID NO.1; the expression cassette of the glucosamine-6-phosphate acetyltransferase gene GNA1 is shown in SEQ ID NO.2; the expression cassette of the N-acetylglucosamine-2-epimerase gene AGE is shown in SEQ ID NO.

3.

13. The recombinant Bacillus subtilis according to claim 12, characterized in that Contain at least one of the following characteristics: (1) When the target gene is the sialic acid synthase gene neuB, the integration site is one or more of the leading strand of the gene ftsY, the leading strand of the gene yocN, the lagging strand of the gene ftsY, and the lagging strand of the gene yneT; (2) When the target gene is a coding gene in the T7 expression system, the integration site is one or more of the lagging strand of gene yabC, the leading strand of gene yhzF, the leading strand of gene ycgP, the lagging strand of gene lytR, the lagging strand of gene yhzF, the lagging strand of gene yqhB, the lagging strand of gene yneT, and the leading strand of gene perR.

14. The method for constructing the recombinant Bacillus subtilis according to any one of claims 10 to 13, comprising the following steps: The Cre recombinase and the target gene expression frame with the restriction site loxP are introduced into the Bacillus subtilis host bacteria, so that the target gene expression frame replaces the gene at the integration site on the host bacteria genome, thereby obtaining the recombinant Bacillus subtilis.

15. Use of the recombinant cell according to claim 8, the recombinant Bacillus subtilis according to any one of claims 10 to 13, or the recombinant Bacillus subtilis obtained by the construction method according to claim 14 in biosynthesis.

16. The use according to claim 15, characterized in that Contain at least one of the following characteristics: (i) The products of synthesis include proteins, amino acids or organic acids; (ii) the products of synthesis include sialic acid; (iii) The synthesized products are regulated by the T7 expression system.

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