Bacillus subtilis recombinant bacterium for expressing cyclic diadenylate and application of bacillus subtilis recombinant bacterium

Abstract

The invention discloses bacillus subtilis recombinant bacteria for expressing cyclic diadenylate and application of the bacillus subtilis recombinant bacteria. A method comprises the following gene modification on bacillus subtilis: (1) knocking out a ykoY gene; and (2) designing an interference RNA sequence of the targeted GdpP gene. GdpP expression can be silenced by knocking out the ykoY gene, interfering RNA further inhibits GdpP activity, and the yield of c-di-AMP is increased to 60 pg / mL or above through dual regulation and control and is increased by 20 times or above compared with that of wild type c-di-AMP.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant Bacillus subtilis strain capable of highly expressing cyclic diadenosine monophosphate (c-di-AMP), a construction method thereof, and an application thereof. Background Art

[0002] Cyclic diadenosine monophosphate (c-di-AMP) is an important second messenger molecule in bacteria, and is involved in regulating physiological functions such as growth, cell wall metabolism, and pathogenicity. The existing technologies for producing c-di-AMP using Bacillus subtilis have the following key defects: (1) Endogenous metabolic regulation is strict and growth inhibition is significant Bacillus subtilis synthesizes c-di-AMP through DacA and maintains dynamic balance through degradation by the GdpP phosphodiesterase. Overexpression of DacA will lead to the accumulation of c-di-AMP, causing osmotic imbalance and abnormal cell wall synthesis, and inhibiting cell growth (as reported in the literature Smith et al., Journal of Bacteriology, 2017, 199(12): e00543-17. Knocking out GdpP can increase the level of c-di-AMP but is accompanied by a slowdown in cell growth).

[0003] Excessive c-di-AMP will interfere with the regulation of sporulation, trigger the sporulation process in advance, and reduce the biomass yield (reference: Gundlach, J., Herzberg, C., Mäder, U., et al. (2015). Cyclic di-AMP Homeostasis in Bacillus subtilis: Both Lack and High Level Accumulation of the Nucleotide Are Detrimental for Cell Growth.).

[0004] (2) Insufficient supply of precursor ATP limits synthesis The ATP regeneration ability of Bacillus subtilis is weaker than that of Escherichia coli, especially under anaerobic conditions, and the synthesis of c-di-AMP consumes 2 molecules of ATP. When highly expressed, it is easy to cause energy depletion and affect cell growth, as described in the literature Zhang et al., Applied and Environmental Microbiology, 2019, 85(14): e00784-19.

[0005] (3) Mainly intracellular accumulation, low secretion and extraction efficiency c-di-AMP is primarily intracellular. While Bacillus subtilis secretes it, it lacks an active efflux mechanism. Traditional methods like ultrasonication can easily lead to product degradation or contamination with impurities (such as DNA and proteins), complicating purification (reference: Gründel, A., Schellack, C., & Harz, H. (2012). Analysis of Cyclic di-AMP in Bacteria.).

[0006] (4) Genetic manipulation and fermentation process challenges The efficiency of homologous recombination in Bacillus subtilis is low, knocking out genes (such as GdpP) requires long fragment homology arm screening, and the plasmid is unstable (easily lost during fermentation).

[0007] During high-density fermentation, bacteria tend to form aggregates, affecting dissolved oxygen transfer and product uniformity. Components such as Mn²⁺ in the culture medium may destroy the stability of c-di-AMP (reference Wahl, A., Hu, J., et al. (2021). Enhanced c-di-AMP Production by Engineered Bacillus subtilis through Fermentation Optimization: The Critical Role of Metal Ions). Summary of the Invention

[0008] To address the shortcomings of the existing technology, the present invention provides a Bacillus subtilis strain that efficiently expresses cyclic di-AMP, as well as a method for constructing and using the strain. By knocking out the ykoY gene of Bacillus subtilis and designing an interfering RNA sequence targeting the GdpP gene, the present invention reduces the efficiency of the GdpP enzyme in Bacillus subtilis in hydrolyzing cyclic di-AMP, thereby increasing the cyclic di-AMP content in Bacillus subtilis.

[0009] The technical solution adopted in the present invention is as follows: In a first aspect of the present invention, a method for constructing a recombinant Bacillus subtilis strain that efficiently expresses cyclic diadenylation is provided, the method comprising genetically modifying the Bacillus subtilis strain as follows: (1) Knockout of the ykoY gene; (2) Design interfering RNA sequences targeting the GdpP gene.

[0010] In one or some embodiments of the present invention, in step (1), the homology arm design includes: upstream homology arm: 500 bp before the ykoY start codon (including the promoter region), downstream homology arm: 300 bp after the ykoY stop codon.

[0011] In one or some embodiments of the present invention, in step (1), the expression vector used is pMUTIN4, and the recombinant plasmid is pMUTIN4-ykoY:ermC.

[0012] In one or some embodiments of the present invention, in step (1), the ermC resistance marker is inserted to replace the ykoY gene.

[0013] In one or some embodiments of the present invention, in step (1), the gene knockout process includes: Electroporation conditions: Preparation of competent cells: Bacillus subtilis strain in the mid-logarithmic phase (OD 600 = 0.6); Electroporation parameters: 2.5 kV, 200 Ω, 25 μF; Temperature screening: Culturing at 42 °C for 12 h to promote plasmid integration; Culturing at 30 °C to screen for single crossover recombinants.

[0014] In the second aspect of the present invention, a recombinant Bacillus subtilis bacterium constructed by the above method is provided.

[0015] In the third aspect of the present invention, an application of the recombinant Bacillus subtilis bacterium in the production of c-di-AMP is provided.

[0016] The application method includes: administering the recombinant Bacillus subtilis bacterium to an animal; specifically, adding the recombinant Bacillus subtilis bacterium to feed or drinking water or premix.

[0017] After the animal consumes the recombinant Bacillus subtilis bacterium, the recombinant Bacillus subtilis bacterium can not be killed by gastric acid, and will eventually reach the intestine through the stomach and express c-di-AMP in the intestine. It has functions such as positive immune activation, inhibition of pathogen colonization, and promotion of probiotic proliferation.

[0018] In the fourth aspect of the present invention, a method for producing c-di-AMP by the recombinant Bacillus subtilis bacterium is provided, and the method includes a process of producing c-di-AMP by an aerobic fermentation process.

[0019] In one or some embodiments of the present invention, the recombinant Bacillus subtilis bacterium is inoculated into an LB medium supplemented with chloramphenicol, and subjected to aerobic fermentation to obtain a fermentation broth containing c-di-AMP. The fermentation broth can be further ultrasonically disrupted to purify c-di-AMP.

[0020] Compared with the related technologies known to the inventors of the present invention, one of the technical solutions of the present invention has the following beneficial effects: Knocking out the ykoY gene can silence the expression of GdpP, and interfering RNA further inhibits the activity of GdpP. The dual regulation increases the c-di-AMP production to more than 60 pg / mL, which is more than 20 times higher than that of the wild type.

[0021] Removing the inhibition of endogenous c-di-AMP metabolism improves the growth state of the bacteria, and the fermentation biomass increases by 30% compared with the control.

[0022] Optimizing the secretion pathway (although there is no active excretion, the increased intracellular accumulation improves the extraction efficiency by 45% after cell disruption). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not unduly limit the invention.

[0024] Figure 1 It is the PCR verification electrophoresis diagram of the ykoY gene knockout strain (wild type: 3.2 kb, knockout strain: 2.1 kb).

[0025] Figure 2 It is the color reaction of the c-di-AMP concentration Elisa experiment.

[0026] Figure 3 It is the standard curve of c-di-AMP concentration (y = 214.7x + 1.3834, R² = 0.9999).

[0027] Figure 4 It is the culture diagram of mouse feces.

[0028] Figure 5 It is the concentration of type I interferon and CXCL10 in mouse blood. Among them, A represents the concentration of type I interferon in mouse blood, and B represents the concentration of CXCL10 in mouse blood. DETAILED DESCRIPTION OF THE INVENTION

[0029] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] Example 1 A method for constructing a recombinant Bacillus subtilis strain that highly expresses cyclic diadenosine monophosphate, using Bacillus subtilis 168 (this strain can be obtained through commercial channels, etc.) as the starting strain, and this method includes the following steps: 1. Technical solution for knocking out the ykoY gene The position of the ykoY gene is at positions 2275200 - 2275697 of GenBank accession number NC_000964.3, and it is on the complementary strand.

[0033] 1) Vector construction Target gene: Design of ykoY homologous arms; Upstream homologous arm: 500 bp (including the promoter region) before the start codon of ykoY; Downstream homologous arm: 300 bp after the stop codon of ykoY; Resistance marker: ermC (erythromycin resistance gene); Recombinant plasmid: pMUTIN4 - ykoY:ermC (based on a temperature - sensitive shuttle vector).

[0034] 2) Gene knockout process Electroporation conditions: Preparation of competent cells: Bacillus subtilis 168 strain in the mid - logarithmic phase (OD 600 = 0.6); Electroporation parameters: 2.5 kV, 200 Ω, 25 μF (0.2 cm electroporation cuvette); Temperature screening: Cultivate at 42 °C for 12 h to promote plasmid integration; Cultivate at 30 °C to screen for single - crossover recombinants; Verification of double - crossover: Use primers F: 5'-ATGGCTGCTGAAGAAG - 3' (as shown in SEQ ID NO: 1) and R: 5'-CTAGCTTCTTCGCTTTC - 3' (as shown in SEQ ID NO: 2) for PCR verification; Expected band: Wild - type 3.2 kb → knockout strain 2.1 kb, as Figure 1 shown.

[0035] 2. Construction of interfering RNA targeting the GdpP gene 1) Determine the GdpP gene sequence Bacillus subtilis GdpP gene (GenBank accession number: NC_000964.3, base positions 1823456 - 1824452).

[0036] 2) Construction of interfering RNA sequence An online tool RNAi Designer was used to design an interfering RNA sequence targeting the GdpP gene. The nucleotide sequence of the interfering RNA is 5'-GCCUACGUUUCGAUCUUGA-3', as shown in SEQ ID NO: 3.

[0037] 3) Construction of a vector expressing interfering RNA The shuttle plasmid pHT315 of Bacillus subtilis was selected. The designed interfering RNA sequence was cloned behind the P43 promoter and the rrnB T1T2 terminator sequence of Bacillus subtilis was used. Through restriction endonuclease digestion and ligation reactions, the interfering RNA sequence was inserted into the vector. The ligation product was transformed into Bacillus subtilis for amplification and verification, and it was confirmed by sequencing that the interfering RNA sequence was correctly inserted into the vector.

[0038] 4) Introduction of the vector into Bacillus subtilis Competent cells were prepared by electroporation method. The constructed vector was transformed into the competent cells, and the transformed cells were spread on a medium containing chloramphenicol (pHT315 carries a chloramphenicol resistance gene) to screen for positive clones containing the vector. The obtained recombinant bacteria were Bacillus subtilis recombinant bacteria ABF-KC60. Two of the recombinant bacteria were selected and named ABF-KC60 clone 1 and ABF-KC60 clone 2 respectively.

[0039] Example 2 ABF-KC60 experiment The ABF-KC60 strain was inoculated into LB medium supplemented with chloramphenicol and cultured at 37°C and 180 rpm for 18 hours. The wild-type Bacillus subtilis 168 was used as a control, and all conditions were the same as those of ABF-KC60 except for the different strains.

[0040] After the culture was completed, the culture broth was ultrasonically disrupted. Four parallel samples were taken from each group and measured using a cyclic diadenosine monophosphate Elisa kit. The grouping is shown in Table 1 below: Table 1 Among them, Control 1-1 to Control 1-4 are wild-type Bacillus subtilis 168, Control 2-1 to Control 2-4 are wild-type Bacillus subtilis PY79 (this strain can be obtained through commercial channels, etc.), Experiments 1-1 to Experiments 1-4 are recombinant bacteria with a single knockout of the ykoy gene, Experiments 2-1 to Experiments 2-4 are recombinant bacteria with a single interference of the GdpP gene, Experiments 3-1 to Experiments 3-4 are ABF-KC60 clone 1 with the ykoy gene knocked out + the GdpP gene interfered in Example 1, and Experiments 4-1 to Experiments 4-4 are ABF-KC60 clone 2 with the ykoy gene knocked out + the GdpP gene interfered in Example 1.

[0041] The experimental color reaction is as Figure 2 shown.

[0042] The grouped readings are as shown in Table 2 below, and the readings in Table 2 correspond to the experiments in Table 1: Table 2 Based on the data of Standard Curve 1-6, a linear regression equation is made as Figure 3 shown.

[0043] Substituting the data into the standard curve, the average concentration of each sample is shown in Table 3: Table 3 Experimental conclusion: Compared with the control group, the recombinant Bacillus subtilis ABF-KC60 of the present invention can highly express cyclic diadenosine monophosphate.

[0044] Example 3 ABF-KC60 in vivo retention experiment The experiment was carried out in three groups. Each group selected 10 healthy female BL / c mice for the experiment, and the mice were intragastrically administered 10^6 CFU of different strains.

[0045] The control group (CG) selected an engineered bacterium of Bacillus subtilis expressing green fluorescent protein (the starting bacterium is Bacillus subtilis 168) to exclude the influence of the strain itself and its secreted products on the experiment except for the target protein; The experimental groups were ABF-KC60 clone 1 (G1) and ABF-KC60 clone 2 (G2) in Example 1 respectively.

[0046] Fecal samples were taken at different time periods and cultured directionally on plates containing antibiotics to determine whether the strain reached the intestine and whether it was still active after passing through the digestive system.

[0047] The experimental results are as Figure 4 shown, and the experiment proves that the strains in the experimental groups can reach the intestine and are active.

[0048] Example 4 Intestinal expression experiment Since the expression level of c-di-AMP in the intestine cannot be directly measured, an indirect measurement method was selected. Two groups of mice were fed with engineered bacteria of Bacillus subtilis that can express green fluorescent protein (the starting strain is Bacillus subtilis 168) and ABF-KC60 respectively. Because cyclic diadenylate is an agonist of the sting protein, the secretion of cyclic diadenylate can be indirectly demonstrated by measuring the blood concentrations of chemotactic factor 10 (CXCL10) and interferon (IFN-Ⅰ).

[0049] The experimental results are as Figure 5 , after 15 days of feeding, the blood concentrations of chemotactic factor 10 ( Figure 5 B in Figure 5 A in and interferon (

[0050] Matters not covered by this invention are well-known technologies.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for constructing a recombinant Bacillus subtilis strain efficiently expressing cyclic diadenosine monophosphate, characterized in that, The method includes the following genetic modifications to Bacillus subtilis: (1) Knock out the ykoY gene; (2) Design an interfering RNA sequence targeting the GdpP gene, and the nucleotide sequence of the interfering RNA is shown as SEQ ID NO:

3.

2. The construction method of the Bacillus subtilis recombinant bacterium for highly expressing cyclic diadenosine monophosphate according to claim 1, characterized in that, In step (1), this step includes the design of homologous arms: upstream homologous arm: 500 bp before the start codon of ykoY, downstream homologous arm: 300 bp after the stop codon of ykoY.

3. The construction method of the Bacillus subtilis recombinant bacterium for highly expressing cyclic di-adenosine monophosphate according to claim 1, characterized in that, In step (1), the expression vector used is pMUTIN4, and the recombinant plasmid is pMUTIN4-ykoY:ermC.

4. The construction method of the recombinant Bacillus subtilis strain for highly expressing cyclic di-adenosine monophosphate according to claim 1, characterized in that, In step (1), the ermC resistance marker is inserted to replace the ykoY gene.

5. The construction method of the Bacillus subtilis recombinant bacterium for highly expressing cyclic diadenosine monophosphate according to claim 1, characterized in that, In step (1), the gene knockout process includes: Electroporation conditions: Competent cell preparation: Bacillus subtilis strain in the mid-logarithmic phase (OD 600 = 0.6); Electroporation parameters: 2.5 kV, 200 Ω, 25 μF; Temperature screening: Cultivate at 42°C for 12 h to promote plasmid integration; Cultivate at 30°C to screen for single crossover recombinants.

6. A recombinant Bacillus subtilis bacterium constructed by the method described in any one of claims 1 to 5.

7. Use of the recombinant Bacillus subtilis bacterium described in claim 6 in the production of c-di-AMP.

8. The application according to claim 7, characterized in that, The application method includes: administering the recombinant Bacillus subtilis bacterium to an animal; specifically, adding the recombinant Bacillus subtilis bacterium to feed or drinking water or premix.

9. A method for producing c-di-AMP by the recombinant Bacillus subtilis strain as claimed in claim 6, characterized in that, The method includes the process of producing c-di-AMP by an aerobic fermentation process.

10. The method for producing c-di-AMP by the recombinant Bacillus subtilis strain according to claim 9, characterized in that, Inoculate the recombinant Bacillus subtilis bacterium into LB medium supplemented with chloramphenicol, perform aerobic fermentation to obtain a fermentation broth containing c-di-AMP; then ultrasonically disrupt the fermentation broth and purify c-di-AMP.

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