Application of c-di-AMP and synthetase coding gene cdaA of c-di-AMP in regulation and control of biosynthesis of streptococcus thermophilus exopolysaccharides

Abstract

The invention discloses c-di-AMP and application of a synthetase coding gene cdaA of the c-di-AMP in regulation and control of biosynthesis of streptococcus thermophilus exopolysaccharides. The unique coding gene of the synthetase of the second messenger molecule c-di-AMP is a cdaA gene, and the sequence of the cdaA gene is as shown in SEQ. ID. NO. 13. The change of the level of intracellular c-di-AMP has a regulation effect on the yield of extracellular polysaccharide EPS; and the increase of the level of c-di-AMP in streptococcus thermophilus can promote the synthesis of extracellular polysaccharide. The invention further provides genetic engineering streptococcus thermophilus overexpressing the cdaA gene and a construction method and application of the genetic engineering streptococcus thermophilus overexpressing the cdaA gene. And constructing a recombinant expression vector of the overexpressed cdaA gene, and transferring the recombinant expression vector into streptococcus thermophilus to obtain the gene engineering streptococcus thermophilus of the overexpressed cdaA gene. The streptococcus thermophilus polysaccharide biosynthesis is changed by adjusting the intracellular c-di-AMP level, and a new strategy and method are provided for metabolic regulation of streptococcus thermophilus.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology and relates to the application of c-di-AMP and its synthase encoding gene cdaA in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus. Background Art

[0002] Streptococcus thermophilus is a representative food-grade fermentation bacteria with excellent fermentation properties such as protein degradation, lactose metabolism and secretion of exopolysaccharides. [1] Streptococcus thermophilus exopolysaccharides (EPS) are a class of high molecular weight polysaccharide compounds secreted outside the cell wall during growth. EPS has stable water retention and gelling properties. It can not only increase the texture of yogurt and cream, but also serve as a thickener and stabilizer to improve the quality of yogurt. In addition, it also has physiological activities such as antibacterial, anti-tumor, antioxidant, and lipid-lowering. [2] .

[0003] The low EPS production rate of Streptococcus thermophilus seriously hinders its industrial application, so there is an urgent need to improve the fine regulation of Streptococcus thermophilus. The second messenger molecule cyclic di-AMP (c-di-AMP) is widely present in Gram-positive bacteria. In the process of rapid response to external environmental signal transduction regulation, it participates in the regulation of physiological functions such as cell growth, biofilm formation, potassium homeostasis, DNA integrity, cell wall synthesis and fatty acid synthesis. [3,4] . Currently, the regulatory function of c-di-AMP has only been found in Bacillus subtilis and Streptococcus mutans, and research on second messenger molecules has not yet been conducted in Streptococcus thermophilus. Although there have been some studies on signal molecules such as the two-component signal transduction system and quorum sensing that Streptococcus thermophilus uses to respond to the environment, the lack of understanding of second messenger molecules has seriously hindered more in-depth regulatory transformation and engineering applications of Streptococcus thermophilus. Therefore, analyzing the metabolic regulatory mechanism of the second messenger molecule c-di-AMP in Streptococcus thermophilus will deepen our understanding of second messenger molecules and lay a theoretical foundation for metabolic regulation and targeted transformation of Streptococcus thermophilus.

[0004] In summary, the EPS yield of Streptococcus thermophilus in the existing technology is low, there is a lack of research on its second messenger molecule c-di-AMP, and the relationship between the second messenger molecule c-di-AMP and EPS synthesis is not clear.

[0005] [1]WAY,CHANYI R M,NGUYEN H T H,et al.Extracellular PolysaccharideExtraction from Streptococcus thermophilus in Fermented Milk[J].MicrobiologySpectrum,2022,10(2):e0228021.

[0006] [2]ZHOU Y,CUI Y,QU X.Comparative transcriptome analysis for thebiosynthesis of antioxidant exopolysaccharide in Streptococcus thermophilusCS6[J].Journal of Science of Food and Agriculture,2022,102(12):5321-32

[0007] [3]KRUGER L,HERZBERG C,WICKE D,et al.Sustained Control of PyruvateCarboxylase by the Essential Second Messenger Cyclic di-AMP in Bacillussubtilis[J].Microbiology,2021,13(1):e0360221.

[0008] [4]XIONG Z Q,FAN Y Z,SONG X,et al.The second messenger c-di-AMPmediates bacterial exopolysaccharide biosynthesis:a review[J].MolecularBiology Reports,2020,47(11):9149-57. Summary of the Invention

[0009] Based on the shortcomings of the above-mentioned prior art, the purpose of this application is to provide the use of c-di-AMP and its synthase encoding gene cdaA in regulating the biosynthesis of exopolysaccharides in Streptococcus thermophilus. The present invention identifies a c-di-AMP synthase encoding gene cdaA in Streptococcus thermophilus Benshit. By measuring the production and molecular weight of exopolysaccharides in cdaA knockout and overexpression strains, the regulatory effect of c-di-AMP on the biosynthesis of exopolysaccharides in Streptococcus thermophilus is demonstrated.

[0010] The present invention provides the following technical solutions:

[0011] One of the technical solutions of the present invention provides the use of the cdaA gene in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus.

[0012] The present invention identifies a c-di-AMP synthase encoding gene cdaA in thermophilic Streptococcus Benshit by LC-MS / MS. The nucleotide sequence of the cdaA gene is shown in SEQ.ID.NO.13.

[0013] A second technical solution of the present invention provides the use of a second messenger molecule, c-di-AMP, in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus. The only gene encoding the synthase of the second messenger molecule, c-di-AMP, is the cdaA gene.

[0014] Furthermore, when the intracellular level of the second messenger molecule c-di-AMP increases in Streptococcus thermophilus, it promotes the biosynthesis of extracellular polysaccharides.

[0015] By measuring the extracellular polysaccharide production of the gene knockout strain, overexpression strain and knockout complement strain, it was found that changes in intracellular c-di-AMP levels have a regulatory effect on the production of extracellular polysaccharides EPS: the decrease in c-di-AMP levels in thermophilic Streptococcus has almost no effect on the production of extracellular polysaccharides, but the increase in intracellular c-di-AMP levels significantly increases the production of extracellular polysaccharides.

[0016] A third technical solution of the present invention provides a method for constructing a genetically engineered Streptococcus thermophilus that overexpresses the cdaA gene. The starting strain of the genetically engineered Streptococcus thermophilus is the Benshit strain of Streptococcus thermophilus, which has a deposit number of CGMCC No. 12098 and was deposited with the General Microbiology Center of the China Culture Collection of Microorganisms on January 22, 2016. It is classified as Streptococcus thermophilus. The cdaA gene is overexpressed in the starting strain.

[0017] Furthermore, the construction method comprises the following steps:

[0018] A recombinant expression vector for overexpressing the cdaA gene is constructed, and the recombinant expression vector is transferred into the thermophilic Streptococcus Benshit strain to obtain a genetically engineered thermophilic Streptococcus overexpressing the cdaA gene.

[0019] A fourth technical solution of the present invention provides a genetically engineered Streptococcus thermophilus that overexpresses the cdaA gene obtained by the above method.

[0020] A fifth technical solution of the present invention provides a use of a genetically engineered thermophilic Streptococcus that overexpresses the cdaA gene in promoting the biosynthesis of exopolysaccharides of the thermophilic Streptococcus.

[0021] A sixth technical solution of the present invention provides a method for preparing flavored fermented milk, comprising:

[0022] The genetically engineered thermophilic streptococcus overexpressing the cdaA gene is inoculated into raw milk, and the flavored fermented milk is obtained after fermentation for a period of time.

[0023] Furthermore, the inoculum size was 1×10 6 ~1×10 8 CFU / mL;

[0024] Furthermore, the fermentation condition is 35-45° C. for 4-24 hours.

[0025] Compared with the prior art, the present invention has at least the following innovations and advantages:

[0026] (1) The present invention identified the c-di-AMP synthase encoding gene in Streptococcus thermophilus by LC-MS / MS, constructed an overexpression strain and a gene deletion strain, and characterized the extracellular polysaccharide production of the strain to determine the regulatory function of c-di-AMP on the EPS of Streptococcus thermophilus.

[0027] (2) The present invention changes the polysaccharide biosynthesis of Streptococcus thermophilus by regulating the intracellular c-di-AMP level, providing a new strategy and method for the metabolic regulation of Streptococcus thermophilus.

[0028] (3) In the prior art, the improvement of the biosynthesis of extracellular polysaccharides of Streptococcus thermophilus is often studied. The present application provides research on improving the biosynthesis of extracellular polysaccharides of Streptococcus thermophilus, which helps to expand the scope of application of Streptococcus thermophilus, optimize production, and promote the innovation of fermented dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the c-di-AMP detection situation in Example 1;

[0030] A: c-di-AMP detection results in c-di-AMP standard, Streptococcus thermophilus Benshit, and the cdaA gene-knockout strain BenshitΔcdaA; B: c-di-AMP detection in Escherichia coli / pET30a-cdaA heterologously expressing c-di-AMP, and c-di-AMP positive and negative controls; C: Mass spectrum of c-di-AMP;

[0031] Figure 2 The results of the analysis of genes encoding DAC domains or DisA_N domains with c-di-AMP synthase activity in the genome of Streptococcus thermophilus in Example 1;

[0032] Figure 3 This is the construction of the cdaA knockout strain in Example 1;

[0033] Among them, A: gene knockout scheme; B: agarose gel electrophoresis screening results of cdaA knockout transformants;

[0034] Figure 4 This is a statistical result diagram of the extracellular polysaccharide production in Example 2;

[0035] Among them, A: detection of extracellular polysaccharide production of gene cdaA knockout strain; B: detection of extracellular polysaccharide production of gene cdaA overexpression strain and complementation strain. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0037] All raw materials in the present invention are not particularly limited in their sources and can be purchased commercially or prepared according to conventional methods known to those skilled in the art. Tryptone and yeast extract powder were purchased from OXOID; soy peptone and beef extract powder were purchased from Beijing Luqiao; lactose, β-glycerophosphate disodium, and lactose were purchased from Shanghai Yuanye; MgSO4·7H2O, glycine, sorbitol, CaCl2, and MgCl2 were purchased from Shenggong; and bacterial agar powder was purchased from Haibo Biological.

[0038] 1) Materials involved in the present invention:

[0039] LM17 medium: tryptone 5g / L, soy peptone 5g / L, yeast extract powder 2.5g / L, beef extract powder 5g / L, lactose 20g / L, β-glycerophosphate disodium 19g / L, MgSO 4. 7 H2O 0.58g / L, lactose 20g / L.

[0040] LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, for culturing E. coli. Add 20 g / L agar powder to the solid medium.

[0041] SLM17 medium: LM17 medium containing 10% (w / v) glycine.

[0042] LM17MC recovery medium: 0.4 mol / L sorbitol, 2 mmol / L CaCl2, and 20 mmol / L MgCl2.

[0043] 2) Instruments and equipment involved in the present invention:

[0044] Plate Smart small plate centrifuge, Spectra Max i3x multifunctional microplate reader, fully automatic growth curve analyzer, S1000 PCR instrument, Power basic electrophoresis instrument, ChemiDoc XRS+ gel imager, Micropulse electroporation converter, SHIMADZU high-performance liquid chromatography triple quadrupole mass spectrometer, and Wyatt Technology high-performance size exclusion chromatography system.

[0045] Example 1 Identification of c-di-AMP synthase gene of Streptococcus thermophilus

[0046] (1) Experimental methods:

[0047] (1) Preparation and transformation of competent Escherichia coli and Streptococcus thermophilus

[0048] Preparation of competent E. coli: Streak the Ecoli.Top10 strain stored in glycerol at -80°C on an LB plate and culture at 37°C for 12-24 hours until the colony reaches a diameter of 1-2 mm. Pick a single colony into a test tube containing 4 mL of LB medium and culture it on a shaker for 12 hours (37°C, 200 rpm). Inoculate it with 1% inoculum into 50 mL of LB medium and culture it on a shaker until the OD 600= 0.3-0.5, collect the cells by centrifugation at 4500 × g for 10 min at 4°C, gently wash the cells with ice-cold 0.1 mol / L CaCl₂ solution, let it stand on ice for 20 min, then centrifuge at 4500 × g for 5 min. Repeat the wash twice. Store in pre-chilled 0.1 mol / L CaCl₂ glycerol buffer in EP tubes, aliquot in 100 μL, and store at -80°C until needed.

[0049] Transformation verification of competent E. coli: Thaw one tube of competent cells on ice, add 1 μL of plasmid or 10 μL of ligation product, and mix gently. Incubate on ice for 30 minutes, incubate at 42°C for 90 seconds, and then incubate on ice for 3 minutes. Add 900 μL of fresh LB liquid medium and incubate on a shaker for 45-60 minutes (37°C, 200 rpm). Centrifuge at 4500 × g for 5 minutes, discard 900 μL of supernatant, and finally spread 100 μL of the bacterial solution on a resistance plate and incubate at 37°C for 24-48 hours. Pick colonies from the resistance plate and add 10 μL of sterile water. After pipetting and mixing, take 0.5 μL of the bacterial solution as a template and perform PCR amplification using the designed verification primers. Detect the size of the PCR product by gel electrophoresis.

[0050] Preparation of competent cells of Streptococcus thermophilus Benshit and plasmid transformation: Streak Benshit onto LM17 plates, culture in a 37°C incubator for 24 h, culture in LM17 medium for 12 h, inoculate 3% (v / v) in LM17 liquid medium, and culture for 3 to 5 h until OD 600 The concentration of 0.4 to 0.6, incubated in LM17 medium containing 10% glycine for 1 hour, placed on ice for 10 minutes, collected bacteria (4 ° C, 5000 rpm, 10 minutes), washed twice with 0.4 mol / L sorbitol and 5mmol / L potassium dihydrogen phosphate solution buffer, took an appropriate amount of pre-cooled glycerol buffer to resuspend, 100 μ L per tube, ready for electrotransformation. 100 ng of plasmid was added to the competent cells and mixed thoroughly, ice bathed for 30 minutes, transferred to a 2 mm electroporation cup cooled on ice, set the electroporation voltage to 2.5 kV, and quickly added 900 μ L of recovery medium (LM17 medium with 0.4 mol / L sorbitol, 2mmol / L CaCl2 and 20mmol / L MgCl2) after electroporation. Transfer to an EP tube and culture at 37 ° C for 4 to 6 hours. After centrifugation, discard 900 μ L of medium and apply 100 μ L of the remaining resistant plate to the plate and culture anaerobically at 37 ° C for 48 hours.

[0051] (2) PCR amplification reaction system

[0052] The PCR amplification system, in a 25 μL reaction volume, consisted of: 12.5 μL 2× Phanta Max Master Mix, 1 μL Primer-F, 1 μL Primer-R, 1 μL DNA template, and the volume was made up with ddH2O. The amplification program was as follows: 95°C for 3 min; 98°C for 10 s; Tm (primer annealing temperature) for 30 s; 72°C for 30 s / kb, 30 cycles; 72°C for 10 min. The amplified product was verified by agarose gel electrophoresis. PCR product recovery: The target fragment, which amplified a single band, was recovered using the Axygen Clean-Up Kit, and the recovered DNA concentration was determined.

[0053] (3) Seamless cloning reaction system

[0054] E. coli containing the plasmid was inoculated into liquid LB medium (the pIB184 backbone is erythromycin (Em)-resistant, and the pKLH353 backbone is chloramphenicol (Cm)-resistant). After incubation at 37°C in a shaker for 12-15 hours, the cells were collected by centrifugation in an EP tube and the plasmid was extracted according to the procedures in the Axygen Plasmid Extraction Kit. The vector DNA was cut with a restriction endonuclease that recognized the internal sequence of the plasmid double-stranded DNA and cut the double strands at specific sites. The cut was performed at 37°C for 3-6 hours. The digested DNA mixture was electrophoresed, and fluorescent bands were observed under UV light to confirm and separate the digested fragments. The vector was recovered using a gel tapping recovery kit, and the recovered DNA concentration was measured. The ligation was completed according to the Clon Express II One-Step Cloning Kit. The molar ratio of vector to target fragment was 1:2. The reaction system consisted of 0.4 pmol of target fragment, 0.2 pmol of vector fragment, 2 μL of 5×CE Buffer (Clon Express buffer), 1 μL of Exnase (a recombinase that has the ability to recognize and bind to DNA fragments with homology arms, mediating the exchange and ligation of DNA chains, thereby achieving seamless ligation between the vector and the target fragment), and ddH2O added up to 10 μL.

[0055] (4) Construction of knockout and overexpression plasmids

[0056] Using the whole genome of Streptococcus thermophilus Benshit as a template, the cdaA gene (the nucleotide sequence of the cdaA gene is shown in SEQ.ID.NO.13 in Table 1) was amplified using primers pET30a-cdaA-F / R (the primer sequences are shown in SEQ.ID.NO.1 to 2 in Table 1) and inserted into the plasmid pET30a digested with restriction endonucleases NdeⅠ and XhoⅠ. After successful verification, the transformant was sent to BGI for sequencing and named pET30a-cdaA. This plasmid is a heterologous expression plasmid for protein purification.

[0057] Using the whole genome of Streptococcus thermophilus Benshit as a template, the cdaA gene was amplified using primers pcdaA-F / R (primer sequences are shown in SEQ.ID.NO.3-4 in Table 1) and inserted into the plasmid pIB184 digested with restriction endonucleases BamHI and EcoRI. After successful transformation, the transformant was sent to BGI for sequencing and named pcdaA. This plasmid is an overexpression plasmid.

[0058] Using the whole genome of Streptococcus thermophilus Benshit as a template, primers TS-11D3DH-696-F1 / R1 (primer sequences are shown in SEQ.ID.NO.5-6 in Table 1) were used to amplify crRNA. Using the whole genome of Streptococcus thermophilus Benshit as a template, primers TS-11D3DH-696-F2 / R2 (primer sequences are shown in SEQ.ID.NO.7-8 in Table 1) and TS-11D3DH-696-F3 / R3 (primer sequences are shown in SEQ.ID.NO.9-10 in Table 1) were used to amplify the upstream and downstream homology arms of cdaA, and plasmid TS-11D3DH-1170 was used as a template, and primers TS-11D3DH-696-F4 / R4 (primer sequences are shown in SEQ.ID.NO.11-12 in Table 1) were used to amplify the vector. The crRNA and upstream and downstream homology arms were connected through overlap PCR and inserted into the reverse amplification vector. After successful transformation, the transformants were sent to BGI for sequencing. The plasmid was named TS-11D3DH-696, which is a gene editing plasmid.

[0059] Table 1 Related sequences of Example 1

[0060]

[0061]

[0062]

[0063] (5) Construction of knockout strains, overexpression strains, and complementation strains

[0064] Plasmid TS-11D3DH-696 was introduced into the Streptococcus thermophilus Benshit strain via electroporation and cultured in a 37°C incubator for 36-48 hours. Successfully transformed colonies were selected and identified using colony PCR and gene sequencing to identify mutant strains with target gene knockout.

[0065] Plasmid pIB184-cdaA was introduced into the thermophilic Streptococcus Benshit strain via electroporation and cultured in a 37°C incubator for 36-48 hours. Successfully transformed colonies were selected and identified using colony PCR and gene sequencing to identify mutant strains that overexpressed the target gene.

[0066] Plasmid pIB184-cdaA was introduced into the thermophilic Streptococcus Benshit△cdaA strain by electroporation and cultured in a 37°C incubator for 36-48 hours. Complementing strains were identified by colony PCR.

[0067] (6)LC-MS / MS

[0068] A Shimadzu LCMC-8060NX high-performance liquid chromatograph coupled with a triple quadrupole mass spectrometer equipped with an SPD detector was used. The detection wavelength was 260 nm, the column temperature was 40°C, and the flow rate was 0.15 mL / min. For the determination of c-di-AMP levels in Streptococcus thermophilus Benshit, mobile phase A was water and mobile phase B was acetonitrile; for the determination of c-di-AMP levels in Escherichia coli, mobile phase A was water and mobile phase B was methanol. Gradient elution was used in both assays. Elution times are shown in Table 2 below.

[0069] Table 2

[0070] Time (min) Mobile phase A% Mobile phase B% 0 95 5 5.0 0 100 5.1 95 5 7 95 5

[0071] (2) Results and Discussion

[0072] The cell lysate of Streptococcus thermophilus Benshit was analyzed by LC-MS ( Figure 1 ), the present application found that the cell lysate and the c-di-AMP standard had significant chromatographic peaks at the same peak time ( Figure 1 A), and after knocking out the cdaA gene, the chromatographic peak disappeared significantly. Mass spectrometry detection of the chromatographic peak components showed ( Figure 1 C), whose mass-to-charge ratio is consistent with that of c-di-AMP standard, mainly manifested as fragment ion peaks of 524.10, 330.20 and 136.10 ( Figure 1 B), indicating that the lysate does contain c-di-AMP, thus confirming that S. thermophilus Benshit has the ability to synthesize c-di-AMP.

[0073] In order to identify the enzyme encoding genes involved in c-di-AMP synthesis in Streptococcus thermophilus, the present application analyzed the genes encoding DAC domain or DisA_N domain with c-di-AMP synthase activity in the genome, such as Figure 2Research has revealed that gene orf0696 encodes a DisA_N domain, suggesting it may be encoding c-di-AMP synthase. The protein encoded by orf0696 contains 236 amino acids (sequence shown in SEQ.ID.NO.14), sharing 47% and 33% amino acid sequence similarity with CdaA and DisA of Bacillus subtilis, respectively. Therefore, gene orf0696 was named cdaA.

[0074] To explore the function of cdaA, the present invention heterologously expressed the cdaA gene in the E. coli strain BL21 (DE3) that is unable to synthesize c-di-AMP. The plasmid pET30a-cdaA was transformed into competent E. coli cells to obtain the E. coli heterologous expression strain BL21 (DE3)-cdaA, and the intracellular c-di-AMP level was detected by LC-MS / MS. The chromatographic peak of c-di-AMP was successfully detected in the lysate of the BL21 (DE3)-cdaA strain ( Figure 1 C), while no c-di-AMP was detected in the lysate of the negative control BL21(DE3), indicating that the gene cdaA is indeed the gene encoding c-di-AMP synthase.

[0075] In addition, the present invention knocks out the gene cdaA ( Figure 3 A), and the intracellular c-di-AMP level was detected by LC-MS / MS. Compared with the Benshit strain, no c-di-AMP was detected in the knockout strain Benshit△cdaA ( Figure 3 B) It was further confirmed that the cdaA gene is the only gene encoding c-di-AMP synthase in Streptococcus thermophilus.

[0076] Example 2: c-di-AMP regulates exopolysaccharide biosynthesis

[0077] (1) Experimental methods:

[0078] Yield determination:

[0079] Pick a single colony of Benshit and inoculate it into 1 mL of LM17 medium. Culture it at 37°C for 10 hours and adjust the OD 600To 1.0. 3% of the seed liquid was inoculated into LM17 liquid culture medium and cultured at 37°C for 24 hours. The fermentation broth was centrifuged at 9000g for 15 minutes at 4°C, the supernatant was taken, and 80% (v / v) trichloroacetic acid TAC solution was added to a final concentration of 4% (v / v) to remove protein. The mixture was allowed to stand at 4°C overnight, centrifuged again at 9000g for 15 minutes at 4°C, and the supernatant was taken. The supernatant was dialyzed with deionized water for two days, the deionized water was replaced every 4 hours, and the volume was fixed for polysaccharide content determination.

[0080] The polysaccharide content was determined using the phenol-sulfuric acid method: 1 mL of the fermentation supernatant was added with 0.5 mL of 3% phenol solution and 5 mL of concentrated sulfuric acid, and the mixture was allowed to stand at room temperature for 20 minutes. The absorbance was then measured at the maximum absorption wavelength λmax = 490 nm, with sterile culture medium as a control.

[0081] The other experimental methods involved in this embodiment are the same as those in Example 1 and will not be repeated here.

[0082] (2) Results and Discussion

[0083] To investigate whether c-di-AMP regulates exopolysaccharide biosynthesis in Streptococcus thermophilus, the cdaA overexpression plasmid pIB184-cdaA was electroporated into S. thermophilus Benshit to construct the c-di-AMP synthase-overexpressing strain Benshit / pcdaA. Exopolysaccharide production was measured in the wild-type strain Benshit, the knockout strain Benshit△cdaA, the overexpressing strain Benshit / pcdaA, and the complemented strain Benshit△cdaA / pcdaA after 24 days of culture in CDM medium.

[0084] like Figure 4 As shown in A, the extracellular polysaccharide production of strains Benshit and Benshit△cdaA was 74.2 mg / L and 68.5 mg / L, respectively. Figure 4 As shown in Figure B, the exopolysaccharide production of strains Benshit / pIB184, Benshit / pcdaA, and Benshit△cdaA / pcdaA was 73.1 mg / L, 95.9 mg / L, and 66.3 mg / L, respectively. The exopolysaccharide production of the cdaA overexpressing strain increased significantly by 31% compared to the control strain Benshit / pIB184. The exopolysaccharide production of the complemented strain Benshit△cdaA / pcdaA was significantly reduced compared to the overexpressing strain Benshit / pcdaA. These results indicate that elevated c-di-AMP levels in Streptococcus thermophilus Benshit promote exopolysaccharide biosynthesis.

[0085] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

[0086] Other sequence information involved in the embodiments of the present invention:

[0087] SEQ.ID.NO.14 (amino acid sequence of the protein encoded by gene orf0696):

[0088] MTLVQGLVLFIFVKIISDFIGFFTTISYLMNQVINYGAIAAVVIFAPELRSALETFGRTPQHFLQNKEVSSDEKLVQAFVKAVKYMSPRKIGALVSIEQTQTLQEQIATGIPLDAVVTG ELLINIFIPNTPLHDGAVIIRDNKVTTACSYLPLSESNKISKEFGTRHRAAIGLSEQTDALTFVVSEETGAISIAYKGNFLHDLSVQEFEHELSLILLKEQEPRQLFFQRWIGGGEK*.

Claims

1. The application of cdaA gene in regulating the biosynthesis of exopolysaccharide of Streptococcus thermophilus, characterized in that: The nucleotide sequence of the cdaA gene is shown in SEQ.ID.NO.

13.

2. The application of the second messenger molecule c-di-AMP in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus is characterized in that: The only gene encoding the synthase of the second messenger molecule c-di-AMP is the cdaA gene as claimed in claim 1; the production of extracellular polysaccharides is increased by increasing the intracellular c-di-AMP level of Streptococcus thermophilus.

3. A method for constructing a genetically engineered Streptococcus thermophilus that overexpresses the cdaA gene, characterized in that: The starting strain of the genetically engineered thermophilic Streptococcus is the thermophilic Streptococcus Benshit strain, the deposit number of the thermophilic Streptococcus Benshit strain is CGMCC No. 12098, and it was deposited in the China General Microbiological Culture Collection Center on January 22, 2016; the gene cdaA is overexpressed in the starting strain.

4. The method for constructing a genetically engineered thermophilic Streptococcus overexpressing the cdaA gene according to claim 3, characterized in that: The following steps are involved: A recombinant expression vector for overexpressing the cdaA gene is constructed, and the recombinant expression vector is transferred into the thermophilic Streptococcus Benshit strain to obtain a genetically engineered thermophilic Streptococcus overexpressing the cdaA gene.

5. A genetically engineered Streptococcus thermophilus overexpressing the cdaA gene obtained by the construction method according to any one of claims 3-4. 6 . Use of the genetically engineered Streptococcus thermophilus overexpressing the cdaA gene as claimed in claim 5 in improving the biosynthesis of exopolysaccharides in Streptococcus thermophilus.

7. A method for preparing flavored fermented milk, characterized in that: The genetically engineered thermophilic Streptococcus overexpressing the cdaA gene as claimed in claim 5 is inoculated into raw milk, and the flavored fermented milk is obtained after fermentation for a period of time.

8. The method for preparing flavored fermented milk according to claim 7, characterized in that: The inoculum size of the genetically engineered thermophilic Streptococcus overexpressing the cdaA gene was 1×10 6 ~1×10 8 CFU / mL.

9. The method for preparing flavored fermented milk according to claim 7, characterized in that: The fermentation temperature is 35-45°C.

10. The method for preparing flavored fermented milk according to claim 7, characterized in that: The fermentation time is 4 to 24 hours.

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