Bacillus subtilis with reduced spontaneous mutation rate and application thereof

Abstract

The invention relates to bacillus subtilis with reduced spontaneous mutation rate and application thereof, and belongs to the technical field of gene engineering. According to the invention, the bacillus subtilis with reduced spontaneous mutation rate is constructed by knocking out genes yolk D, yozK and yozL for coding potential error-prone DNA polymerase in a bacillus subtilis genome under the condition that the specific growth rate of the strain is not influenced, and the bacillus subtilis can be applied to improving the expression stability of free or integrated expression exogenous genes. The strain is used as a chassis strain, and an N-acetylneuraminic acid synthesis pathway is constructed in an integrated expression manner, so that N-acetylneuraminic acid can still be efficiently produced after 59 generations of passage. The strain is used as a chassis strain to construct plasmids for expressing fluorescence reporter genes, and the correct replication of genes on the plasmids can be ensured to exceed 47 generations under the condition that the plasmids are not lost. The bacillus subtilis provided by the invention provides an efficient universal tool for improving genetic stability, and can be applied to construction of cell factories with stable expression.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a Bacillus subtilis with reduced spontaneous mutation rate and application thereof. Background Art

[0002] Advances in synthetic biology and metabolic engineering technologies have provided new approaches for the design and construction of microbial cell factories, which are essential for green biomanufacturing. However, the lack of stability in bioprocesses limits their application on an industrial scale. This low stability is usually caused by population heterogeneity, including phenotypic variation and genetic mutations, leading to the emergence of non-productive or low-producing cells. These cells are easily enriched during fermentation due to their light metabolic burden and fast growth, thus affecting the stability of the bioprocess. Compared with phenotypic heterogeneity, genetic heterogeneity is more harmful because it can lead to irreversible loss of production capacity. Therefore, improving genetic stability is crucial to enhancing the robustness of bioprocesses. 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 in vitro assembly of multi-fragment DNA, and by constructing a mutant library encoding the pathway, genetically stable pathway construction is screened out; population quality control and product addiction systems rely on biosensors that respond to target products or intermediates, and give production or high-yield cell growth advantages through growth and synthesis coupling, and enrich production cells or high-yield cell subpopulations during fermentation to improve the stability of biological processes. However, these methods all have their shortcomings. For example, large-scale combinatorial pathway methods rely on efficient in vitro assembly of multi-fragment DNA, which is only applicable to genes encoded on plasmids, and the process of screening genetically stable pathway construction is complicated and time-consuming; population quality control and product addiction systems rely on biosensors that respond to intermediates. 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 its universality. Therefore, how to develop efficient and universal strategies to reduce genetic heterogeneity and improve production stability is a key issue that needs to be solved urgently. Summary of the invention

[0004] The present invention uses the Cre / lox system to knock out the genes yolD, yozK and yozL encoding potential error-prone DNA polymerase in the Bacillus subtilis genome, thereby constructing a Bacillus subtilis strain with a reduced spontaneous mutation rate without affecting the strain specific growth rate. The strain can be used to improve the expression stability of free or integrated expressed exogenous genes.

[0005] The first object of the present invention is to provide a method for reducing the spontaneous mutation rate of Bacillus subtilis, and to knock out the DNA polymerase encoding genes yolD, yozK and yozL in the genome of Bacillus subtilis.

[0006] Furthermore, the nucleotide sequence of the yolD is shown in SEQ ID NO.1, the nucleotide sequence of the yozK is shown in SEQ ID NO.2, and the nucleotide sequence of the yozL is shown in SEQ ID NO.3.

[0007] The second object of the present invention is to provide a Bacillus subtilis with a reduced spontaneous mutation rate, wherein the DNA polymerase encoding genes yolD, yozK and yozL are knocked out in the genome of the Bacillus subtilis.

[0008] The third object of the present invention is to provide the use of the above-mentioned Bacillus subtilis in improving the expression stability of foreign genes.

[0009] In one embodiment of the present invention, the exogenous gene is the fluorescent reporter gene GFP.

[0010] Furthermore, the expression is free expression or integrated expression.

[0011] Furthermore, the episomal expression step includes transferring the plasmid into the Bacillus subtilis to express the foreign gene.

[0012] Furthermore, the plasmid uses pHT01 or pP43NMK as a backbone.

[0013] In one embodiment of the present invention, the plasmid is based on pHT01 and includes a promoter P veg , used to express the fluorescent reporter gene GFP.

[0014] In one embodiment of the present invention, the plasmid is based on pP43NMK as the backbone, including the promoter P m4 , used to express the fluorescent reporter gene GFP.

[0015] The fourth object of the present invention is to provide the use of the above-mentioned Bacillus subtilis in producing N-acetylneuraminic acid.

[0016] Furthermore, using the Bacillus subtilis as a chassis strain, genes related to the N-acetylglucosamine degradation pathway, genes related to the acetate synthesis pathway, genes related to the lactate synthesis pathway, and genes related to the phosphoenolpyruvate transport pathway are knocked out in the genome, and genes related to the N-acetylneuraminic acid synthesis pathway and sialic acid synthase encoding genes are integrated and expressed.

[0017] Furthermore, the genes related to the N-acetylneuraminic acid synthesis pathway include glmS, GNA1 and AGE.

[0018] Furthermore, the sialic acid synthase encoding gene is neuB.

[0019] In one embodiment of the present invention, the glmS, GNA1 and AGE are overexpressed in multiple copies.

[0020] Furthermore, the nucleotide sequence of the glmS expression cassette is shown in SEQ ID NO.4, the nucleotide sequence of the GNA1 expression cassette is shown in SEQ ID NO.5, and the nucleotide sequence of the AGE expression cassette is shown in SEQ ID NO.6. The above expression cassettes all include a promoter, an RBS sequence and a target gene.

[0021] In one embodiment of the present invention, P xpaC The promoter expresses the neuB, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO.7. The expression cassette includes a promoter, an RBS sequence and a target gene.

[0022] The fifth object of the present invention is to provide a method for improving the stability of N-acetylneuraminic acid produced by Bacillus subtilis, by knocking out the DNA polymerase encoding genes yolD, yozK and yozL in the genome of Bacillus subtilis producing N-acetylneuraminic acid.

[0023] Beneficial effects of the present invention:

[0024] The present invention knocks out potential error-prone DNA polymerase sites in the Bacillus subtilis genome without affecting the growth and growth rate of the strain. Using the Bacillus subtilis provided by the present invention as a chassis strain, an N-acetylneuraminic acid synthesis pathway is constructed by integrating expression, so that it can still efficiently produce N-acetylneuraminic acid after 59 generations of passage, while the control strain can only maintain high yield for 48 generations. Using the bacteria as a chassis strain, a plasmid expressing the fluorescent reporter gene GFP with a strong combinatorial promoter is constructed, which can ensure that the genes on the plasmid are correctly replicated for more than 47 generations without losing the plasmid, while the control strain can only ensure that the plasmid genes are correctly replicated for 34 generations. The Bacillus subtilis provided by the present invention provides an efficient and universal tool for improving genetic stability, which can be applied to construct a cell factory for stable expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 is the spontaneous mutation rate of Bacillus subtilis after knocking out different genes in Example 1 of the present invention;

[0027] Figure 2 is the specific growth rate of Bacillus subtilis after knocking out different genes in Example 1 of the present invention;

[0028] Figure 3 is the spontaneous mutation rate of Bacillus subtilis after knocking out different genes in combination in Example 1 of the present invention;

[0029] Figure 4 is the specific growth rate of Bacillus subtilis after knocking out different genes in combination in Example 1 of the present invention;

[0030] Figure 5 The invention relates to the use of Bacillus subtilis with a low spontaneous mutation rate in the production of N-acetylneuraminic acid in Example 2, wherein a is a schematic diagram of the synthesis pathway of N-acetylneuraminic acid, b is the change in the yield of N-acetylneuraminic acid after passage of different lineages of the C1 strain, c is the specific growth rate after passage of different lineages of the C1 strain, d is a comparison of the yield of N-acetylneuraminic acid between the C1 strain and the C1ΔDKL strain, e is a comparison of the spontaneous mutation rates of the C1 strain and the C1ΔDKL strain, and f is a change in the yield of N-acetylneuraminic acid after passage of the C1 strain and the C1ΔDKL strain;

[0031] Figure 6 The present invention shows an application of Bacillus subtilis with a low spontaneous mutation rate in improving the stability of plasmid expression in Example 3, wherein a is a schematic diagram of the structure of H-veg-GFP and N-m4-GFP recombinant plasmids, b is a comparison of the fluorescence intensities of BS168 (H-veg-GFP) strain and ΔDKL (H-veg-GFP) strain after subculture, and c is a comparison of the fluorescence intensities of BS168 (N-m4-GFP) strain and ΔDKL (N-m4-GFP) strain after subculture. DETAILED DESCRIPTION

[0032] 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.

[0033] The materials used in the following examples are as follows:

[0034] DNA polymerase was purchased from Takara, PCR product nucleic acid purification kit was purchased from Thermo Scientific, seamless cloning kit used for plasmid construction was purchased from Wuhan Abotek Biotechnology, and plasmid extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0035] 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.

[0036] The N-acetylneuraminic acid production stability analysis 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 / L MgSO4·7H2O, 6 g / L urea and 60 g / L glucose, pH = 7.0.

[0037] SM medium: 200 mL 5x SM salts, 10 mL 100x trace elements, 10 mL 100x iron-citrate solution, 10 mL glucose (50%), 10 mL tryptophan (5 mg / mL), add sodium L-glutamate (40%) to a final concentration of 1%, dilute to 1000 ml with ddH2O, sterilize at 121°C for 20 min;

[0038] 5x SM salt solution: 175 g K2HPO4, 75 g KH2PO4, 12.5 g Na3-Citrate·2H2O, 2.5 g MgSO4·7H2O, dilute to 2000 mL with ddH2O, sterilize at 121°C for 20 min;

[0039] 10mL 100x trace elements: 0.55g CaCl2, 0.735g CaCl2·2H2O, 0.1gMnCl2·4H2O, 0.17gZnCl2, 0.033g CuCl2·2H2O, 0.043g CuCl2·6H2O, 0.06gCoCl2·6H2O, 0.06g Na2MoO4·2H2O, dilute to 1000mL with ddH2O, filter and sterilize with 0.22μm sterile filter;

[0040] 100x iron-citrate solution: 0.0135 g FeCl3·6H2O, 0.1 g Na3-Citrate·3H2O, dilute to 100 mL with ddH2O, and filter through a 0.22 μm sterile filter to sterilize.

[0041] 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.

[0042] The Bacillus subtilis transformation method used in the following examples is based on the literature Anagnostopoulos, C., Spizizen, J., 1961. Requirements for transformation in Bacillus subtilis. Journal of bacteriology 81, 741-746. and Zhang, X.-Z., Zhang, Y.-HP, 2011. Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial biotechnology 4, 98-105.

[0043] The Bacillus subtilis gene knockout method used in the following examples is referenced in 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.

[0044] The mutation rate analysis method used in the following examples is based on Sankar, TS, Wastuwidyaningtyas, BD, Dong, Y., Lewis, SA, Wang, JD, 2016. The nature of mutations induced by replication-transcription collisions. Nature 535, 178-181., as shown below:

[0045] Streak the strain of interest onto LB agar plates. After overnight culture, inoculate a single colony into a 15 mL shake tube filled with LB and incubate for 5 hours. Then inoculate the culture into a 15 mL shake tube filled with fresh SM medium at a ratio of 2%. Grow until OD 600= 0.4-0.6, dilute the culture to 1×10 -5 Then, 200 μL of the cells were aliquoted into 96-well plates and cultured at 37°C until OD 600 =0.4-0.6. In each biological replicate, each strain should have at least 18 wells of 200 μL parallel culture. 200 μL of culture was spread on selective medium (SM agar plate supplemented with 5 mg / L trimethoprim) and cultured at 45°C. After culturing for 48 hours and 72 hours, the number of trimethoprim-resistant mutants was counted and recorded. At the same time, the four cultures were gradiently diluted and spread on complete medium (SM agar plate), and the number of colonies was counted and recorded. Then, the mutation rate per cell per generation was calculated using the fluctuation analysis calculator (FALCOR) web tool. The thyA gene 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.

[0046] The primer sequences involved in the following examples are as follows:

[0047] Table 1 Primer sequences, templates and uses

[0048]

[0049]

[0050]

[0051] Example 1: Construction of low mutation rate Bacillus subtilis

[0052] 1. Single deletion (primer sequences are shown in Table 1)

[0053] The prophage region includes many genes of unknown function, which may include genes encoding error-prone DNA polymerases. To analyze the effect of knocking out the prophage region on the spontaneous mutation rate, six prophage regions were deleted to construct strain Δ6, because the phage region includes many genes of unknown function. Non-homologous end joining is a low-fidelity DNA double-strand break repair pathway, so the genes ykoV and ligD encoding the Bacillus subtilis non-homologous end joining system were knocked out to construct strain ΔNHEJ. Genes encoding error-prone DNA polymerases that have been experimentally proven in Bacillus subtilis include yqjH, yqjW, and uvrX. The three genes were knocked out in the genome of Bacillus subtilis to obtain strains ΔyqjH, ΔyqjW, and ΔuvrX, respectively. There are also some genes encoding potential error-prone DNA polymerases in Bacillus subtilis, such as yqjX, yolD, yozK and yozL. The functional studies of these potential error-prone DNA polymerases are mainly based on bioinformatics analysis rather than experimental verification. Four genes were knocked out in the Bacillus subtilis genome to obtain strains ΔyqjX, ΔyolD, ΔyozK and ΔyozL, respectively.

[0054] The mutation rate analysis of the 9 different recombinant Bacillus subtilis constructed above was performed, and the unmodified Bacillus subtilis BS168 was used as the control. The mutation rate analysis results are shown in Figure 1 As shown, deletion of the phage region and the gene encoding the non-homologous end joining system are not suitable for the development of low mutation rate chassis cells. The spontaneous mutation rate of Bacillus subtilis was not significantly changed after knocking out the relevant genes. Compared with BS168, the spontaneous mutation rate of ΔNHEJ was even increased. Only knocking out yqjW can reduce the spontaneous mutation rate by 15.4%, single knocking out yqjH will lead to an increase in the mutation rate, while single knocking out uvrX does not significantly change the spontaneous mutation rate. Single knocking out yolD, yozK and yozL can reduce the mutation rate by 21.3%, 19.4% and 19.6%, respectively, which also provides an experimental basis for the functional study of these potential error-prone DNA polymerases.

[0055] In addition, the effects of single knockout of these unstable elements on cell growth were analyzed. The results of the specific growth rate test of recombinant Bacillus subtilis are as follows: Figure 2 As shown, no growth effects were observed.

[0056] 2. Combination deletion (primer sequences are shown in Table 1)

[0057] Since the mutual influence between error-prone DNA polymerases is still unclear, the effect of combinatorial deletion of error-prone DNA polymerase genes on the spontaneous mutation rate was then analyzed. The strain ΔDU was obtained by simultaneous knockout of yolD and uvrX, the strain ΔDK was obtained by simultaneous knockout of yolD and yozK, the strain ΔKL was obtained by simultaneous knockout of yozK and yozL, and the strain ΔDL was obtained by simultaneous knockout of yolD, yozK, and yozL. The strain ΔDKL was obtained by simultaneous knockout of yolD, yozK, yozL, and uvrX. The strain ΔDKLU was obtained by simultaneous knockout of yolD, yozK, yozL, and yqjW. The strain ΔDKLW was obtained by simultaneous knockout of yolD, yozK, yozL, uvrX, and yqjW.

[0058] The mutation rate analysis of the eight different recombinant Bacillus subtilis constructed above was performed, with the unmodified Bacillus subtilis BS168 as the control. The mutation rate analysis results are shown in Figure 3 As shown in the figure, the spontaneous mutation rate of ΔDU is 29.5% lower than that of BS168. Therefore, the simultaneous knockout of yolD and uvrX is beneficial for constructing low mutation rate chassis cells. Compared with Bacillus subtilis 168, the spontaneous mutation rates of ΔDL strain and ΔDKL strain were reduced by 34.4% and 41.8%, respectively. Single knockout of uvrX and yqjW in strain ΔDKL will lead to a slight increase in mutation rate, while the simultaneous knockout of yqjW and uvrX in strain ΔDKL will lead to a significant increase in mutation rate, even exceeding the mutation rate of Bacillus subtilis 168. This may be due to the simultaneous deletion of these five genes inducing other mutagenic mechanisms. In summary, strain ΔDKL with simultaneous deletion of yolD, yozK and yozL can be used as a low mutation rate chassis cell, wherein the nucleotide sequence of yolD is shown in SEQ ID NO.1, the nucleotide sequence of yozK is shown in SEQ ID NO.2, and the nucleotide sequence of yozL is shown in SEQ ID NO.3.

[0059] In addition, the effects of knocking out these unstable elements on cell growth were analyzed. The results of the specific growth rate test of the recombinant Bacillus subtilis were as follows: Figure 4 As shown, no growth effects were observed.

[0060] Example 2: Low mutation rate Bacillus subtilis improves the stability of N-acetylneuraminic acid production

[0061] N-acetylneuraminic acid (NeuAc), 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.

[0062] A knockout strain of N-acetylglucosamine degradation pathway genes gamA (Gene ID: 938425), nagA (Gene ID: 936621), nagB (Gene ID: 936619), acetate synthesis pathway gene ackA (Gene ID: 937347), lactate synthesis pathway gene ldh (Gene ID: 938348) and phosphoenolpyruvate transport pathway gene ptsG (Gene ID: 939255) was used, and N-acetylneuraminic acid pathway genes glmS (sequence as shown in SEQ ID NO.4, the sequence shown includes a promoter and RBS sequence), GNA1 (sequence as shown in SEQ ID NO.5, the sequence shown includes a promoter and RBS sequence) and AGE (sequence as shown in SEQ ID NO.6, the sequence shown includes a promoter and RBS sequence) were enhanced in a multi-copy form, and P xpaC The Bacillus subtilis strain with the promoter-enhanced gene neuB encoding sialic acid synthase (sequence shown in SEQ ID NO.7, which includes the promoter and RBS sequence) was used as the starting strain and named C1. The N-acetylneuraminic acid synthesis pathway is as follows Figure 5 a. The stability of NeuAc production in C1 was evaluated by analyzing the production performance of different cell division cultures. The results showed that the NeuAc production of each lineage of C1 strain gradually decreased after 48 generations ( Figure 5 b). Correspondingly, the specific growth rate also gradually increases ( Figure 5 c).

[0063] The genes yolD, yozK and yozL in the C1 genome were knocked out to construct strain C1ΔDKL. Compared with C1, the spontaneous mutation rate of C1ΔDKL was reduced by 54.3%, while the growth and NeuAc production were not affected ( Figure 5 d and Figure 5 e) Then, industrial scale-up is simulated by serial passage, e.g. Figure 5As shown in (f), C1ΔDKL exhibited more stable NeuAc production and could stably maintain high-level NeuAc production for up to 59 generations, which was 1.23 times higher than that of C1.

[0064] The specific steps of the method of continuous passage simulating industrial scale are as follows:

[0065] Continuous subculture simulates industrial-scale fermentation: Continuous subculture can simulate the industrial scale-up process. By monitoring the production performance of the culture during the subculture process, the production stability of the strain can be evaluated. The CI and C1ΔDKL strains were inoculated on LB agar plates. After overnight culture, a single colony was inoculated into a 250 mL shake flask containing 25 mL of NeuAc fermentation medium and cultured at 37 ° C and 220 rpm for 23 hours. The culture was then inoculated into fresh fermentation medium with an inoculum size of 2% and cultured at 37 ° C and 220 rpm 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 passages and culture storage, all glycerol stocks were inoculated into 24-well deep-well plates containing 1.5 mL of fermentation medium with an inoculation volume of 1%. After 72 hours of cultivation, the culture was harvested to determine NeuAc production.

[0066] Example 3: Low mutation rate Bacillus subtilis improves the replication fidelity of plasmid expression system

[0067] Plasmid expression systems are widely used because of their ease of operation and multi-copy characteristics. Plasmid loss is currently a major problem for the stability of plasmid expression systems. Plasmids and host genomes share a set of DNA replication and repair systems, so in addition to plasmid loss, plasmid expression systems are also affected by host mutations. Therefore, plasmid loss and mutation are two completely different concepts. Due to the multi-copy characteristics of plasmids, the relevant genes expressed on plasmids often impose metabolic burdens on the host and affect growth. Therefore, loss-of-function mutations of relevant genes on plasmids can be enriched by conferring growth advantages to mutants, thereby compromising the stability of plasmid expression systems in industrial-scale fermentation. Therefore, high-fidelity replication of plasmid expression systems is very important for stable production. There are many strategies to solve the problem of plasmid loss, such as antibiotic supplementation and nutritional deficiency complementation. However, people pay little attention to mutations of relevant genes on plasmids. Since plasmids and host genomes share a common set of DNA replication and repair systems, it is speculated that low-mutation rate chassis cells help improve the genetic stability of relevant genes on plasmid expression systems.

[0068] Using the seamless cloning kit, P vegregulated GFP (sequence as shown in SEQ ID NO.8, the sequence includes promoter and RBS sequence) and P M4 The regulated GFP (sequence is shown in SEQ ID NO.9, and the sequence shown includes the promoter and RBS sequence) was constructed on plasmids pHT01 and pP43NMK, respectively, to construct plasmids H-veg-GFP and N-m4-GFP, and then these two overexpression plasmids were transformed into Bacillus subtilis 168 and ΔDKL with yolD, yozK and yozL knocked out, respectively, to obtain strains 168 (H-veg-GFP), ΔDKL (H-veg-GFP), 168 (N-m4-GFP) and ΔDKL (N-m4-GFP), which were continuously passaged in shake flasks, and the stability of the plasmid expression system was evaluated by analyzing the GFP protein production capacity (fluorescence intensity) of the cultures that had divided the strain for different times during the continuous passage. In order to avoid the interference of plasmid loss, passage and GFP fluorescence analysis were both carried out in LB medium containing the corresponding antibiotics. Figure 6 b and Figure 6 As shown in Figure c, the low mutation rate chassis ΔDKL significantly improved the production stability of GFP on plasmids pHT01 and pP43NMK. The strains ΔDKL (H-veg-GFP) and ΔDKL (N-m4-GFP) could stably and efficiently produce GFP until the 47th generation, while the control strains 168 (H-veg-GFP) and 168 (N-m4-GFP) could only stably and efficiently produce GFP until the 34th generation. This shows that low mutation rate chassis cells have a wide range of application prospects, not only can improve the production stability of small molecule metabolites, but also improve the production stability of proteins in plasmid expression systems.

[0069] The specific steps of continuous passage are as follows:

[0070] To test the stability of the plasmid expression system, a single colony cultured overnight on the LB agar plate was inoculated into a 250 mL shake flask containing 25 mL LB and cultured at 37 °C and 220 rpm for 11 hours. Then, subculture was performed at a 2% inoculum size, 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 .

[0071] 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 reducing the spontaneous mutation rate of Bacillus subtilis, characterized in that: Knockout the DNA polymerase encoding genes yolD, yozK and yozL in the Bacillus subtilis genome.

2. A Bacillus subtilis with reduced spontaneous mutation rate, characterized in that: The DNA polymerase encoding genes yolD, yozK and yozL are knocked out in the genome of the Bacillus subtilis.

3. Use of the Bacillus subtilis according to claim 2 in improving the expression stability of foreign genes.

4. The use according to claim 3, characterized in that: The expression is episomal expression or integrated expression.

5. The use according to claim 4, characterized in that: The episomal expression step includes transferring the plasmid into the Bacillus subtilis to express the foreign gene.

6. The use according to claim 5, characterized in that: The plasmid uses pHT01 or pP43NMK as the backbone.

7. Use of the Bacillus subtilis according to claim 2 in producing N-acetylneuraminic acid.

8. The use according to claim 7, characterized in that: The Bacillus subtilis is used as a chassis strain, genes related to the N-acetylglucosamine degradation pathway, genes related to the acetic acid synthesis pathway, genes related to the lactate synthesis pathway and genes related to the phosphoenolpyruvate transport pathway are knocked out in the genome, and genes related to the N-acetylneuraminic acid synthesis pathway and sialic acid synthase encoding genes are integrated and expressed.

9. The use according to claim 8, characterized in that: The genes related to the N-acetylneuraminic acid synthesis pathway include glmS, GNA1 and AGE.

10. A method for improving the stability of N-acetylneuraminic acid produced by Bacillus subtilis, characterized in that: The DNA polymerase encoding genes yolD, yozK and yozL were knocked out in the genome of Bacillus subtilis producing N-acetylneuraminic acid.

Images