Guided glycosyl transferase EpsE for regulating and controlling streptococcus thermophilus exopolysaccharide biosynthesis and application of guided glycosyl transferase EpsE

Abstract

The invention relates to guided glycosyl transferase EpsE for regulating and controlling streptococcus thermophilus exopolysaccharide biosynthesis and application of the guided glycosyl transferase EpsE. The guide glycosyl transferase gene epsE is located on an eps gene cluster of streptococcus thermophilus Benshit, and the nucleotide sequence of the guide glycosyl transferase gene epsE is as shown in SEQ. ID. No. 1. The knockout of the epsE gene causes the streptococcus thermophilus to completely lose the exopolysaccharide (EPS) generation capability. The streptococcus thermophilus of overexpression guide glycosyl transferase EpsE does not change the monosaccharide type of EPS, and the content of galactose in exopolysaccharides is increased. Synthesis of streptococcus thermophilus EPS is a complex process, and glycosyl transferase EpsE is guided to play a key starting or guiding role in the process. The monosaccharide composition of the EPS is researched and optimized through gene knockout and overexpression, theoretical guidance can be provided for producing the EPS through streptococcus thermophilus in the industry, the development of a more efficient fermentation process is facilitated, the production cost is reduced, and the product quality is improved.

Description

Technical Field

[0001] This application belongs to the field of microbial technology and relates to a guiding glycosyltransferase EpsE for regulating the biosynthesis of exopolysaccharides by Streptococcus thermophilus and its application. Background Art

[0002] Streptococcus thermophilus is widely used in the fermented dairy products and pharmaceutical industries [1] . During growth and metabolism, Streptococcus thermophilus can produce exopolysaccharides (EPS), which usually have food physical and chemical properties such as water retention and viscosity, as well as biological activities such as anti-inflammatory, antioxidant, and antiviral activities. [2–4] EPS biosynthesis mainly involves four stages: the synthesis of nucleotide sugar precursors, the synthesis of oligosaccharide repeating units, polymerization, and export. [5] Among them, the synthesis of oligosaccharide repeating units is initiated by glycosyltransferase (GTF) catalyzing the attachment of nucleotide sugar precursors to lipid carriers, and its expression is directly related to the number and composition of monosaccharides in the repeating units of the corresponding EPS. [6,7] .

[0003] A total of 118 GTFs have been found in 51 known strains of Streptococcus thermophilus [8] , but GTF has substrate specificity, and the gtf genes have high variability. At present, the functions of only a few GTFs have been verified, and the product annotation of gtf genes still poses certain challenges.

[0004] In summary, although Streptococcus thermophilus is widely used in the fermented dairy products and pharmaceutical industries and can produce valuable EPS, there are still many deficiencies in GTF-related research, which makes it difficult to deeply understand the EPS synthesis mechanism and greatly limits the efficient utilization of Streptococcus thermophilus and the research and development optimization of related products.

[0005] [1]Uriot, O., Denis, S., Junjua, M., Roussel, Y., Dary-Mourot, A., & Blanquet-Diot, S. (2017). Streptococcus thermophilus: From yogurt starter to a new promising probiotic candidate?. Journal of Functional Foods, 37, 74–89.

[0006] [2]Che, H., Zhang, H., Tian, Y., Lai, P. F., Xia, Y., Wang, S., & Ai, L. (2019). Exopolysaccharide from Streptococcus thermophilus as stabilizer in fermented dairy: Binding kinetics and interactions with casein of milk. International Journal of Biological Macromolecules, 140, 1018–1025.

[0007] [3]Junjua, M., Kechaou, N., Chain, F., Awussi, A. A., Roussel, Y., Perrin, C., Roux, E., Langella, P., Bermúdez-Humarán, L. G., Roux, Y. L., Chatel, J. M., & Dary-Mourot, A. (2016). A large scale in vitro screening of Streptococcus thermophilus strains revealed strains with a high anti-inflammatory potential. LWT, 70, 78–87.

[0008] [4]Zhang, J., Cao, Y., Wang, J., Guo, X., Zheng, Y., Zhao, W., Mei, X., Guo, T., & Yang, Z. (2016). Physicochemical characteristics and bioactivities of the exopolysaccharide and its sulphated polymer from Streptococcus thermophilus GST-6. Carbohydrate Polymers, 146, 368–375.

[0009] [5]Cui, Y., Jiang, X., Hao, M., Qu, X., & Hu, T. (2017). New advances in exopolysaccharides production of Streptococcus thermophilus. Archives of Microbiology, 199(6), 799–809.

[0010] [6]Van Kranenburg, R., van Swam, I. I., Marugg, J. D., Kleerebezem, M., & de Vos, W. M. (1999b). Exopolysaccharide biosynthesis in Lactococcus lactis NIZOB40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone. Journal of Bacteriology, 181(1), 338–340.

[0011] [7]Van Kranenburg, R., Vos, H. R., van Swam, I. I., Kleerebezem, M., & de Vos, W. M. (1999a). Functional analysis of glycosyltransferase genes from Lactococcus lactis and other gram - positive cocci: Complementation, expression, and diversity. Journal of Bacteriology, 181(20), 6347–6353.

[0012] [8]Ren W, Xia Y J, Wang G Q, et al. Bioactive exopolysaccharides from a S. thermophilus strain: Screening, purification and characterization[J]. International Journal of Biological Macromolecules, 2016(86):402 - 407. SUMMARY OF THE INVENTION

[0013] Based on the disadvantages existing in the above-mentioned prior art, the object of the present application is to provide a guiding glycosyltransferase EpsE for regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus and its application.

[0014] In the bacterial EPS biosynthesis pathway, the guiding glycosyltransferase EpsE participates in the initiation stage or plays a guiding role in the subsequent glycosyl transfer process. The guiding glycosyltransferase EpsE may be responsible for recognizing specific initial receptor molecules (such as lipid carriers, etc.) and catalyzing the transfer of the first nucleotide sugar to the lipid carrier to form an initial glycosylated structure, laying the foundation for the subsequent addition of sugars by other glycosyltransferases and being crucial for the synthesis of EPS repeating units. However, currently, little is known about the glycosyltransferases related to Streptococcus thermophilus, and there is a lack of direct evidence related to EPS biosynthesis.

[0015] In the present application, epsE represents the gene, and EpsE represents its corresponding enzyme (protein); S-3OEepsE represents overexpression of the epsE gene in the Benshit strain, where OE is the abbreviation of Over expression, and the meanings of other expressions are similar; S-3ΔepsE represents knockout of the epsE gene in the Benshit strain, where Δ represents knockout, and the meanings of other expressions are similar. The above representation methods are well-known to those skilled in the art.

[0016] Based on the above object, the present invention provides the following technical solutions:

[0017] One of the technical solutions of the present invention is to provide the application of the guiding glycosyltransferase gene epsE in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus.

[0018] Furthermore, the present invention identifies a guiding glycosyltransferase gene epsE located on the eps gene cluster of Streptococcus thermophilus Benshit (classified as Streptococcus thermophilus, used as the wild-type strain, and the genetically engineered bacterium after transformation is called the S-3 strain) through genomics and homology alignment. The nucleotide sequence of the guiding glycosyltransferase gene epsE is shown in SEQ.ID.No.1.

[0019] Another technical solution of the present invention is to provide the application of the guiding glycosyltransferase EpsE in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus, and the amino acid sequence of the guiding glycosyltransferase EpsE is shown in SEQ.ID.No.2.

[0020] Furthermore, in vitro enzyme activity was verified by bioinformatics and ELISA. The results showed that EpsE had both UDP-galactose and UDP-glucose catalytic activities, and the C-terminal transmembrane region of EpsE was not its active center region. In addition, high-performance liquid chromatography and thin-layer chromatography were used to further quantify the enzyme activity of EpsE, demonstrating that the catalytic activity of EpsE towards UDP-galactose was 1.86 times that of UDP-glucose.

[0021] The third technical solution of the present invention provides a genetically engineered Streptococcus thermophilus overexpressing the gene epsE. The starting strain of the genetically engineered Streptococcus thermophilus overexpressing the gene epsE is Streptococcus thermophilus BENSHIT strain. The preservation number of the Benshit strain is CGMCC 12098. This strain was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on January 22, 2016, and was classified and named Streptococcus thermophilus; the gene epsE was overexpressed in the starting strain.

[0022] The fourth technical solution of the present invention provides a method for constructing a genetically engineered Streptococcus thermophilus overexpressing the gene epsE, and the method includes the following steps:

[0023] Construct a recombinant expression vector for overexpressing the glycosyltransferase epsE gene, and transfer the recombinant expression vector into Streptococcus thermophilus to obtain a genetically engineered Streptococcus thermophilus overexpressing the gene epsE.

[0024] The fifth technical solution of the present invention provides the application of a genetically engineered Streptococcus thermophilus overexpressing the gene epsE in increasing the extracellular polysaccharide yield.

[0025] Furthermore, the monosaccharide composition of the extracellular polysaccharide is galactosamine, galactose and glucose; overexpressing the epsE gene does not change the monosaccharide type of EPS; the genetically engineered Streptococcus thermophilus overexpressing the gene epsE is used to increase the content of galactose in the extracellular polysaccharide. After overexpressing the epsE gene, the monosaccharide composition of EPS changes from galactosamine:galactose:glucose = 1.00:2.94:0.85 of the Streptococcus thermophilus Benshit wild type to galactosamine:galactose:glucose = 1.00:3.43:1.04 of the Streptococcus thermophilus S-3OEepsE overexpression strain.

[0026] The sixth technical solution of the present invention provides a method for preparing dairy products with a high galactose content, using a genetically engineered Streptococcus thermophilus overexpressing the gene epsE for fermentation to increase the galactose content and improve the flavor of dairy products.

[0027] Furthermore, the fermentation conditions are a temperature of 35 to 45 °C and a time of 4 to 24 h; preferably 37 °C, with static fermentation for 24 h.

[0028] Furthermore, the dosage of the genetically engineered Streptococcus thermophilus overexpressing the epsE gene is the viable cell count: 1×10 6 ~1×10 8 CFU / mL; preferably 10 7 CFU / mL.

[0029] The seventh technical solution of the present invention provides a genetically engineered Streptococcus thermophilus with the epsE gene knocked out.

[0030] Furthermore, knocking out the epsE gene results in the complete loss of EPS production ability of Streptococcus thermophilus.

[0031] Furthermore, the knocking out of the epsE gene is carried out by the CRISPR-Cas9 gene editing system. The CRISPR-Cas9 gene editing system is a highly efficient and accurate gene modification tool. It uses CRISPR RNA (crRNA) and transcription activator-like effector nuclease (Cas9 enzyme) to identify and cut specific DNA sequences, and then the cell's DNA repair mechanism (such as non-homologous end joining or homologous directed repair) may lead to the knockout of the target gene.

[0032] Furthermore, the construction method of the genetically engineered Streptococcus thermophilus with the epsE gene knocked out is specifically as follows:

[0033] Construct a CRISPR-Cas9 gene editing plasmid for knocking out the epsE gene, and transfer the gene editing plasmid into Streptococcus thermophilus to obtain the genetically engineered Streptococcus thermophilus with the epsE gene knocked out.

[0034] Furthermore, a method for preparing sugar-free / low-sugar dairy products is provided. This method uses the genetically engineered Streptococcus thermophilus with the epsE gene knocked out for fermentation to reduce the sugar content of dairy products and improve the flavor of dairy products.

[0035] Furthermore, the starting strain and fermentation conditions of the genetically engineered Streptococcus thermophilus with the epsE gene knocked out are the same as those of the genetically engineered Streptococcus thermophilus overexpressing the epsE gene described above.

[0036] Compared with the prior art, the present invention has at least the following innovative points and advantages:

[0037] (1) The guiding glycosyltransferase EpsE provided by this application is related to the synthesis of exopolysaccharide (EPS) of Streptococcus thermophilus. Knocking out the epsE gene results in Streptococcus thermophilus completely losing the ability to produce EPS; overexpressing the epsE gene does not change the monosaccharide types of EPS, and significantly increases the content of galactose in its exopolysaccharide.

[0038] (2) The synthesis of exopolysaccharide (EPS) by Streptococcus thermophilus is a complex process, and the guiding glycosyltransferase EpsE plays a key starting or guiding role in it. Through heterologous gene expression and protein function research, the specific function and role link of EpsE in the EPS synthesis pathway can be clarified, the molecular mechanism of EPS synthesis can be deeply understood, providing an important basis for the basic theoretical research of microbial polysaccharide synthesis, and providing a new perspective for enriching the knowledge system of microbial metabolic regulation network.

[0039] (3) Optimizing the monosaccharide composition of EPS through gene knockout and overexpression research can provide theoretical guidance for the industrial production of EPS by Streptococcus thermophilus, contribute to the development of more efficient fermentation processes, reduce production costs, and improve product quality. In microbial metabolic engineering, the regulation of intermediate metabolic pathways is usually concerned, and targeting EpsE for regulation from the starting step of synthesis provides new strategies and ideas for optimizing microbial metabolic pathways and increasing the yield of target products, with certain innovation and foresight. Description of the Drawings

[0040] Figure 1 For the homology alignment of the guiding glycosyltransferase;

[0041] Reference Signs: A: Amino acid sequence alignment of the guiding glycosyltransferase; among them, the amino acid sequences are respectively from: 1. S. thermophilus Benshit, 2. S. thermophilus CNRZ1066 (AAV62661), 3. S. thermophilus Sfi6 (AAC44012), 4. Lc. lactis NIZO B40 (AAC45231), 5. Xcc strain B100 (CAP51064), 6. S. thermophilus Sfi39 (AAK61899), and the conserved amino acid residues and similar amino acid residues are represented by asterisks and dots respectively; B: Characterization analysis of the guiding glycosyltransferase;

[0042] Figure 2 For the fermentation characteristics of Streptococcus thermophilus Benshit and its engineered strains (S-3OEepsE, S-3ΔepsE);

[0043] Reference Signs: A: Cell growth (OD 600nm); B: Change in total carbohydrate content (total sugar) over time;

[0044] Figure 3 Effect of epsE on EPS chemical composition and molecular characteristics;

[0045] Reference numerals: A: HPSEC-MALS detection map of EPS; B: HPAEC-PAD detection map of EPS; C: Chemical composition and molecular parameters of EPS;

[0046] Figure 4 Expression, purification and verification of catalytic activity of EpsE recombinant protein;

[0047] Reference numerals: A: Construction of heterologous expression plasmid (epsEΔC is truncated C-terminal transmembrane region); B: Protein expression induced by IPTG; C: Purification process of truncated EpsE; D: Purification result of EpsE; E: ELISA verification of in vitro catalytic activity of protein;

[0048] Figure 5 In vitro enzymatic activity of EpsE recombinant protein;

[0049] Reference numerals: A: TLC map; B: HPLC map; C: Quantitative determination of enzyme activity based on HPLC. Detailed implementation manners

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be 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 do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0051] For all raw materials of the present invention, there is no special limitation on their sources, and those purchased on the market or prepared according to the conventional methods well-known to those skilled in the art are all acceptable.

[0052] (I) Materials and reagents

[0053] LM17 medium: Tryptone 5 g / L, Soytone 5 g / L, Yeast extract powder 2.5 g / L, Beef extract powder 5 g / L, β-glycerophosphate disodium 19 g / L, Magnesium sulfate heptahydrate 0.25 g / L, Lactose 20 g / L; Solid medium added with agar powder 20 g / L.

[0054] SLM17 medium: LM17 medium containing 10% glycine.

[0055] LM17MC Recovery Medium: LM17 medium containing 0.4 mol / L sorbitol, 2 mmol / L calcium chloride, and 20 mmol / L magnesium chloride.

[0056] LB Medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, used for culturing Escherichia coli. 20 g / L agar powder is added for solid medium.

[0057] CDM Medium: 10 g / L lactose, 1 g / L sodium acetate, 6 g / L ammonium citrate, 3 g / L KH2PO4, 2.5 g / L K2HPO4, 0.2 g / L MgSO4, 0.05 g / L CaCl2·2H2O, 0.46 g / L aspartic acid, 0.35 g / L asparagine, 0.40 g / L glutamic acid, 0.39 g / L glutamine, 0.44 g / L lysine, 0.13 g / L arginine, 0.15 g / L histidine, 0.68 g / L proline, 0.28 g / L phenylalanine, 0.05 g / L tryptophan, 0.13 g / L methionine, 0.24 g / L alanine, 0.33 g / L valine, 0.48 g / L leucine, 0.22 g / L isoleucine, 0.18 g / L glycine, 0.34 g / L serine, 0.23 g / L threonine, 0.25 g / L cysteine, 0.29 g / L tyrosine, 0.5 g / L ascorbic acid, 0.01 g / L ρ-aminobenzoic acid, 0.01 g / L biotin, 0.005 g / L orotic acid, 0.005 g / L inosine, 0.005 g / L pyridoxamine hydrochloride, DL - 0.0025 g / L 6,8 - lipoic acid, 0.002 g / L pyridoxine hydrochloride, 0.001 g / L nicotinic acid, 0.001 g / L riboflavin, 0.001 g / L calcium pantothenate, 0.001 g / L thiamine HCl, 0.001 g / L folic acid, 0.001 g / L cyanocobalamin.

[0058] Electroporation Buffer: 0.4 mol / L sorbitol, 5 mmol / L KH2PO4 solution (pH 4.5).

[0059] PBS Buffer Solution (10×): 80 mM Na2HPO4, 1.36 M NaCl, 20 mM KH2PO4, 26 mM KCl, pH 7.4.

[0060] NPI - 5: 20 mM Tris - HCl, 500 mM NaCl, 5 mM imidazole, pH 8.0.

[0061] NPI - 20: 20 mM Tris - HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0.

[0062] NPI-100: 20 mM Tris-HCl, 500 mM NaCl, 100 mM imidazole, pH 8.0.

[0063] NPI-500: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0.

[0064] Urea5, Urea20, Urea100, Urea500 are NPI-5, NPI-20, NPI-100, NPI-500 respectively added with 8 M urea.

[0065] (II) Instruments and equipment:

[0066] Plate Smart benchtop centrifuge, Spectra Max i3x multi-functional microplate reader, automatic growth curve analyzer, ultra-micro nucleic acid and protein quantifier Nanodrop 2000, S1000 type PCR instrument, Power basic type electrophoresis instrument, ChemiDoc XRS+ gel imager, HPSEC-MALS multi-angle laser light scattering instrument, high performance anion exchange chromatography tandem pulsed amperometric detector (HPAEC-PAD) system, high performance liquid chromatography system (HPLC).

[0067] Example 1 Identification of the guiding glycosyltransferase gene by homology alignment

[0068] In this example, BLAST was used to perform homology alignment on genomic data to determine the guiding glycosyltransferase involved in EPS synthesis in Streptococcus thermophilus Benshit. The amino acid sequences of AAV62661, AAC44012, AAC45231, CAP51064, and AAK61899 in the figure can be obtained from the platform NCBI familiar to those skilled in the art; the amino acid sequence of S. thermophilus Benshit is shown as SEQ.ID.No.2.

[0069] The amino acid sequence similarity of EpsE of Streptococcus thermophilus Benshit with AAV62661 (S. thermophilus CNRZ1066) and AAC44012 (S. thermophilus Sfi6) is greater than 99%. Among them, AAV62661 is a predicted exopolysaccharide synthesis-related protein of S. thermophilus CNRZ1066, with galactosyltransferase activity, and AAC44012 is a predicted phosphogalactosyltransferase of S. thermophilus Sfi6, guiding the biosynthesis of its EPS repeat unit. Although AAC45231, CAP51064, and AAK61899 have different sources, they are all identified as undecaprenyl phosphate glycosyl-1-phosphate transferases, catalyzing the transfer of the glycosyl-1-phosphate group to undecaprenyl phosphate, and the amino acid sequence similarity of EpsE with the three is greater than 32%( Figure 1 ). Therefore, it is speculated that epsE is the guiding glycosyltransferase gene for EPS biosynthesis in the Benshit strain.

[0070] Example 2 Effects of epsE on cell growth and total sugar content

[0071] To study the effects of epsE on the growth of Benshit cells and EPS synthesis, the epsE knockout strain S-3ΔepsE and the epsE overexpression strain S-3OEepsE were constructed and obtained using technologies such as CRISPR-Cas9. The construction methods of the two strains are as follows:

[0072] (1) PCR amplification reaction system

[0073] PCR reaction system (25 μL): 12.5 μL 2×Phanta Max Master Mix, 1 μL primer-F, 1 μL primer-R, 1 μL DNA template, and ddH2O. PCR amplification program: 95°C for 3 min; 98°C for 10 s; Tm (primer annealing temperature) for 30 s; 72°C for 30 s / kb, cycle 30 times; 72°C for 10 min. (This PCR system and program are applicable to all the following experiments. Just select the template and primers according to the corresponding experimental content.) The amplification products were verified by agarose gel electrophoresis. PCR product recovery: The target fragment with a single band amplified was recovered according to the Axygen clean recovery kit, and the DNA concentration after recovery was measured.

[0074] (2) Seamless cloning reaction system

[0075] The target fragment amplified by PCR and the linearized vector pIB184 were recovered using a gel extraction kit, and the DNA concentration after recovery was measured using a NanoDrop 2000 ultra-micro nucleic acid and protein quantifier. Ligation was completed according to the steps of the Clon Express II One Step Cloning Kit, with a molar ratio of vector to target fragment of 1:2. The 10 μL reaction system included 0.4 pmol of the target fragment, 0.2 pmol of the vector fragment, 2 μL of 5×CEⅡ Buffer, 1 μL of Exnase, and ddH2O.

[0076] (3) Construction of the epsE knockout plasmid:

[0077] Using the Benshit genome as a template, the upstream and downstream homologous arms of epsE were amplified separately using the primers epsE-up-F / R (SEQ.ID.No.3 and SEQ.ID.No.4) and epsE-down-F / R (SEQ.ID.No.5 and SEQ.ID.No.6). Using pKLH356 as a template, epsE_sgRNA was amplified using the primers gRNA-epsA-F / gRNA-R (SEQ.ID.No.7 and SEQ.ID.No.8) and gRNA-epsA-F1 / gRNA-R (SEQ.ID.No.9 and SEQ.ID.No.8). The three fragments were ligated by overlap PCR and inserted into pKLH353 (double digested with XhoI and ClaI), and then transferred into Escherichia coli Top10 competent cells. The plasmid pKLH353-ΔepsE was obtained by resistance screening. It was introduced into Benshit competent cells by electroporation, and the mutant strain S-3ΔepsE was obtained by resistance screening.

[0078] (4) Construction of the epsE overexpression plasmid:

[0079] Using the Benshit genome as a template, the epsE fragment was amplified using the primers epsE-pIB-F1 / R1 (SEQ.ID.No.10 and SEQ.ID.No.11), inserted into pKLH32 (double digested with BamHI and EcoRI), and then transferred into Escherichia coli Top10 competent cells. The plasmid pKLH32-OEepsE was obtained by resistance screening. It was introduced into Benshit competent cells by electroporation, and the mutant strain S-3OEepsE was obtained by resistance screening.

[0080] (5) Preparation and transformation of Escherichia coli competent cells

[0081] Preparation of competent cells: Pick a single colony of E. coli Top10 or E. coli BL21(DE3) and inoculate it into 4 mL of LB medium. Incubate on a shaker for 12 h (37 °C, 200 rpm). Inoculate into 50 mL of LB medium at an inoculation amount of 1%, and incubate on a shaker until the OD 600 reaches 0.3 - 0.5. Centrifuge (4 °C, 4500 rpm, 10 min) to collect the cells. Wash the cells with 0.1 mol / L CaCl2 solution and then resuspend the cells in 1 mL of 0.1 mol / L CaCl2 solution containing 15% glycerol. Aliquot 100 μL into each tube and store at -80 °C for later use.

[0082] Transformation experiment: Mix the competent cells with 10 μL of the ligation product and incubate on ice for 30 min. Heat shock at 42 °C for 90 s, then add 900 μL of LB medium and incubate on a shaker for 60 min (37 °C, 200 rpm). Centrifuge (4500 rpm, 5 min) to collect the cells and spread them on the resistant plate. Incubate at 37 °C to obtain clones. Verify the product size by PCR amplification.

[0083] (6) Preparation and transformation of Streptococcus thermophilus competent cells

[0084] Preparation of competent cells: Pick a single colony of Streptococcus thermophilus Benshit and inoculate it into 15 mL of LM17 medium. Incubate statically at 37 °C for 12 h. Inoculate into 50 mL of LM17 medium at an inoculation amount of 3% and incubate statically at 37 °C until the OD600 reaches 0.3 - 0.5. Mix with an equal volume of SLM17 medium and continue to incubate for 1 h. Let it stand on ice for 10 min and then centrifuge (4 °C, 4000 g, 10 min) to collect the cells. Wash the cells with the electroporation buffer and then resuspend the cells in 1 mL of electroporation buffer containing 15% glycerol. Aliquot 100 μL into each tube and store at -80 °C for later use.

[0085] Electroporation experiment: Mix the competent cells with the knockout plasmid or overexpression plasmid and incubate on ice for 30 min. Transfer to a 2 mm electroporation cuvette and perform electroporation (12.5 kV / cm). Add 900 μL of LM17MC and incubate statically at 37 °C for 6 h. Centrifuge to collect the cells and spread them on the resistant plate. Incubate at 37 °C for 48 h to obtain clones. Verify the product size by PCR amplification.

[0086] The above steps (1) - (6) may also be involved in other embodiments. The subsequent embodiments can refer to the descriptions in this embodiment and will not be elaborated further.

[0087] (7) Experimental results:

[0088] Compared with the Benshit wild type, overexpression and knockout of epsE had no significant effect on cell growth. After knockout of epsE, the strain lost its ability to suspend, while the Benshit wild type and S-3OEepsE were suspended in the culture medium in the early stage, which was conducive to contacting and utilizing fresh culture medium ( Figure 2 A).

[0089] The changes in the total soluble sugar content in the fermentation broth during the culture process were measured. During the culture period of 0 to 8 hours, the strain consumed the lactose in the culture medium and the total sugar content decreased. After 8 hours, the total sugar content of S-3ΔepsE continued to decrease. At this time, the cell growth entered the stable phase and the lactose consumption rate decreased accordingly. However, the total sugar content of Benshit wild type and S-3OEepsE increased after 8 hours, indicating that the bacteria synthesized polysaccharides and secreted them into the culture medium, and the accumulation of EPS lasted until 24 hours or even longer ( Figure 2 B). Therefore, epsE is closely related to EPS biosynthesis. The loss of epsE may affect EPS synthesis, thereby causing the strain to lose its ability to suspend in the culture medium. For facultative anaerobic thermophilic Streptococcus, static culture conditions are not conducive to the growth of the strain and its ability to resist adverse conditions.

[0090] Example 3 Effect of epsE on EPS biosynthesis

[0091] To further explore the effect of epsE on EPS biosynthesis, the wild-type strain of Benshit, S-3ΔepsE and S-3OEepsE were fermented in CDM medium, EPS was extracted, and the differences in molecular mass and monosaccharide composition were detected by HPSEC-MALS (high performance size exclusion chromatography-multi-angle laser light scattering) and HPAEC-PAD (high performance anion exchange chromatography-pulsed amperometric detection).

[0092] The extraction and detection methods of EPS are as follows:

[0093] Thermophilic Streptococcus Benshit and its mutant strains S-3ΔepsE and S-3OEepsE were inoculated into LM17 medium and cultured at 37°C for 12 hours as seed liquid. 1L CDM medium was inoculated with 3% (v / v) inoculation amount of bacteria and cultured at 37°C for 24 hours. After fermentation, the fermentation liquid was vigorously stirred with a glass rod for 30 seconds, and the fermentation liquid was boiled in water for 10 minutes to inactivate the enzyme and most of the bacteria. After cooling to room temperature, centrifugation (10,000×g, 20 minutes, 4°C) was performed to remove cells and coagulated proteins, and dialyzed for 2 days with a dialysis bag with a molecular weight cutoff of 8 to 14 kDa. The supernatant was freeze-dried after centrifugation to obtain EPS: S-3 EPS 、S-3ΔepsE EPS and S-3OEepsE EPS .

[0094] The total sugar content was detected by the phenol-sulfuric acid method; the protein content was detected by the Coomassie brilliant blue method; the molecular weight was detected by high performance size exclusion chromatography-multi-angle light scattering (HPSEC-MALS); the monosaccharide composition was detected by high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD).

[0095] As Figure 3 shown, the results indicated that S-3ΔepsE did not produce EPS at all, while S-3OEepsE still had the ability to generate EPS ( Figure 3 A). The EPS of Benshit and S-3OEepsE were labeled as S-3 EPS and S-3OEepsE EPS respectively. The yield of S-3OEepsE EPS was 156 mg / L, the weight-average molecular weight (Mw) was 6×10 5 Da, and the conformational parameter α was 0.716, indicating that S-3OEepsE EPS showed a random coil structure in solution. Compared with S-3 EPS , there were no obvious differences in its molecular characteristics ( Figure 3 C). Although both S-3 EPS and S-3OEepsE EPS were composed of galactosamine, galactose and glucose, the proportion of galactose in S-3OEepsE EPS increased significantly (from 2.94 to 3.43, Figure 3 B and C). The increase in hydrophilic galactose led to the formation of more hydrogen bonds between EPS molecules or between EPS and water molecules, which might result in an increase in the viscosity and solubility of EPS.

[0096] Example 4 Mechanism of epsE affecting EPS biosynthesis

[0097] To study the mechanism of epsE regulating EPS biosynthesis, the EpsE protein was heterologously expressed and purified, and its enzymatic activity was verified by in vitro reactions.

[0098] (1) Construction of heterologous expression strains:

[0099] Using the genome of Streptococcus thermophilus Benshit as a template, the target genes epsE and epsEΔC were amplified using primers epsE-His-F / R (SEQ.ID.No.12 and SEQ.ID.No.13) and epsEΔC-His-F / R (SEQ.ID.No.14 and SEQ.ID.No.15). After verifying the correct bands by agarose gel electrophoresis, purification and recovery were performed. The target genes and the pET30a plasmid were digested with restriction enzymes Nde I and Xho I respectively to obtain linear vectors. After seamless cloning and ligation of the target genes and the linearized vectors, the products were transferred into Escherichia coli Top10 competent cells, and the plasmids pET30a-epsE and pET30a-epsEΔC were screened by resistance. They were introduced into Escherichia coli BL21(DE3) competent cells, and heterologous expression strains were obtained by resistance screening.

[0100] (2) The methods for protein expression and purification were as follows:

[0101] The plasmids pET30a-epsE and pET30a-epsEΔC were respectively transferred into Escherichia coli BL21(DE3) competent cells, and correct single colonies were picked into 4 mL of LB medium for activation for 12 h. Inoculate into LB liquid medium at a ratio of 3%, and culture in a shaker at 37 °C until OD 600 was approximately 0.6, add IPTG with a final concentration of 0.5 mmol / L, and induce at 16 °C for 18 h. Centrifuge (4 °C, 8000 rpm, 10 min) to collect the bacterial cells, wash twice with PBS buffer and resuspend. Break the cells on ice with an ultrasonic disruptor (working power 800 W, working for 5 s, pausing for 5 s). Centrifuge (4 °C, 8000 rpm, 10 min) to separate the precipitate and the supernatant. Take the original bacterial liquid, the supernatant and the precipitate (PBS resuspension) after ultrasonic disruption respectively. After treatment with the loading buffer, observe the protein expression by 12.5% SDS-PAGE.

[0102] Take an appropriate amount of Ni 2+ -NTA agarose and pack it into an affinity chromatography column, and equilibrate the Ni 2+ column with 5 times the volume of NPI-5 buffer. Filter the supernatant after cell disruption through a 0.45 μm syringe filter and load it onto the column at a flow rate of 10 times the column volume / h. Elute the miscellaneous proteins with 5 times the column volume of NPI-20 and NPI-100 in sequence, and elute and collect the target protein with 10 times the column volume of NPI-500. Concentrate the protein by ultrafiltration centrifugation (10 kDa, 4 °C, 5000 g). Resuspend the precipitate after cell disruption in Urea5, purify it by the above method (replace the buffer with the Urea series), and renature it by dialysis (4 °C, 8 - 14 kDa).

[0103] (3) In vitro enzyme activity detection

[0104] Prepare the reaction system: 8 μg of protein, 2.5 μL of the lipid carrier undecaprenyl phosphate (C55P) (10 mg / mL), 2 μL of DMSO, 6 μL of 10% (v / v) Triton X-100, optionally add 1 μL of cardiolipin (10 mg / mL), 5 μL of Tris-HCl (1 mol / L, pH 7.5), 1 μL of magnesium chloride (1 mol / L), and optionally add 15 μL of UDP-galactose / UDP-glucose / UDP-galactosamine (10 mg / mL). After reacting at 30 °C for 16 h, add an equal volume of chloroform:methanol (1:1) to terminate the reaction, separate the aqueous and organic phases by an extraction reagent (a mixture of 1.5 mL of chloroform, 25 mL of methanol, 23.5 mL of water, and 0.183 g of potassium chloride), and wash and extract the reaction products (uridine monophosphate UMP and lipid-linked precursor respectively).

[0105] Qualitatively refer to the instructions of the uridine monophosphate ELISA detection kit;

[0106] Qualitatively analyze the lipid-linked precursor by thin-layer chromatography TLC: Dissolve the reaction product in 10 μL of chloroform:methanol (1:1) solution, spot it on a silica gel GF254 plate, the developing agent is chloroform:methanol:water:ammonia water (888:4:10:1), and develop the color with iodine vapor;

[0107] Quantitatively analyze the enzyme activity by high-performance liquid chromatography HPLC: Mobile phase A is a mixed solution of 100 mmol / L K2HPO4, 100 mmol / L KH2PO4, and 8 mmol / L tetrabutylammonium hydrogen sulfate, mobile phase B is a mixed solution of 70% A and 30% methanol, XSelectPeptide CSH C18 (4.6 mm × 150 mm) chromatographic column, flow rate 0.5 mL / min, the gradient program is 0 - 2.5 min → 100% A, 2.5 - 16.5 min → 0 - 40% B, 16.5 - 17.5 min → 40 - 100% B, 17.5 - 23.5 min → 100% B, 23.5 - 24.5 min → 100 - 0% B, 24.5 - 32.8 min → 100% A.

[0108] Bioinformatics analysis found that the protein structure of EpsE contains a transmembrane region of 35 - 57 amino acids. Therefore, heterologous expression plasmids of full-length and truncated transmembrane region EpsE were constructed ( Figure 4 A). Protein expression was induced by IPTG. The results showed that truncated EpsE was expressed as inclusion body protein, while EpsE was expressed as both soluble and inclusion body proteins ( Figure 4 B). Truncated EpsE was purified by the denaturation-renaturation method ( Figure 4 C), soluble full-length EpsE was collected, and the protein concentration obtained after concentration was 0.5 mg / mL ( Figure 4D). The reaction product UMP was detected by an ELISA kit. The results showed that both EpsE and truncated EpsE could catalyze UDP-galactose and UDP-glucose, but did not react with UDP-galactosamine ( Figure 4 E). Therefore, the transmembrane region of EpsE is mainly responsible for anchoring the protein to the cell membrane and is not its bioactive site.

[0109] The enzymatic activity of EpsE was further verified by TLC and HPLC. Consistent with the ELISA results, purified EpsE could catalyze the transfer of UDP-galactose and UDP-glucose to the lipid carrier C 55 P in vitro, generating C 55 PP-galactose or C 55 PP-glucose, but did not react with UDP-galactosamine ( Figure 5 A). The relative enzyme activity was quantified by detecting UMP by HPLC. The addition of cardiolipin (DOPG) led to a 1.83-fold increase in UMP production, indicating that DOPG could significantly enhance the catalytic activity of EpsE. In addition, during the in vitro reaction, the catalytic activity of EpsE towards UDP-galactose was 1.86 times that towards UDP-glucose, indicating that although EpsE could bind both nucleotide sugars, there was substrate preference. Figure 5 B and C).

[0110] In summary, EpsE is the initiating glycosyltransferase of Streptococcus thermophilus Benshit, attaching UDP-galactose or UDP-glucose to the lipid carrier C 55 P to initiate the biosynthesis of EPS. Specific EPS-producing strains can be obtained by mutating epsE. For example, EPS-deficient strains and galactose-increased EPS-producing strains can be obtained by knocking out and overexpressing epsE, respectively. The present invention provides a biological modification idea for the production of EPS with specific structures and functions.

[0111] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. Obviously, those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Application of the glycosyltransferase gene epsE in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus, characterized in that: The guiding glycosyltransferase gene epsE is located on the eps gene cluster of thermophilic Streptococcus Benshit, and the nucleotide sequence of the guiding glycosyltransferase gene epsE is shown in SEQ.ID.No.

1.

2. Application of guiding glycosyltransferase EpsE in regulating the biosynthesis of exopolysaccharides of Streptococcus thermophilus, characterized in that: The amino acid sequence of the guide glycosyltransferase EpsE is shown in SEQ.ID.No.

2.

3. A genetically engineered thermophilic Streptococcus overexpressing the gene epsE, characterized in that: The nucleotide sequence of the gene epsE is shown in SEQ.ID.No.

1.

4. The method for constructing a genetically engineered thermophilic Streptococcus overexpressing the gene epsE as claimed in claim 3, characterized in that: The starting strain of the genetically engineered thermophilic Streptococcus that overexpresses the gene epsE 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 General Microbiology Center of the China Microbiological Culture Collection Administration on January 22, 2016; the gene epsE is overexpressed in the starting strain.

5. The construction method according to claim 4, characterized in that: The method specifically comprises: constructing a recombinant expression vector for overexpressing the epsE gene that guides glycosyl transfer, and transferring the recombinant expression vector into thermophilic streptococci to obtain genetically engineered thermophilic streptococci that overexpress the gene epsE.

6. The use of a genetically engineered thermophilic Streptococcus overexpressing the gene epsE as claimed in claim 3 in increasing the production of exopolysaccharides, characterized in that: The monosaccharide composition of the extracellular polysaccharide is galactosamine, galactose and glucose; the genetically engineered thermophilic streptococcus overexpressing the gene epsE is used to increase the content of galactose in the extracellular polysaccharide.

7. A method for preparing a dairy product with a high galactose content, characterized in that: Fermentation was performed using genetically engineered Streptococcus thermophilus that overexpressed the gene epsE to increase the galactose content.

8. The method for preparing a dairy product with a high galactose content according to claim 7, characterized in that: The fermentation conditions are a temperature of 35 to 45° C. and a time of 4 to 24 hours.

9. The method for preparing a dairy product with a high galactose content according to claim 7, characterized in that: The amount of the genetically engineered thermophilic streptococcus overexpressing the gene epsE is: viable bacteria number: 1×10 6 ~1×108CFU / mL.

10. A genetically engineered Streptococcus thermophilus with epsE gene knocked out, characterized in that: The genetically engineered thermophilic streptococcus with epsE gene knocked out completely loses the ability to produce EPS; the method for constructing the genetically engineered thermophilic streptococcus with epsE gene knocked out is specifically as follows: constructing a CRISPR-Cas9 gene editing plasmid with epsE gene knocked out, and transferring the gene editing plasmid into thermophilic streptococcus to obtain the genetically engineered thermophilic streptococcus with epsE gene knocked out; The genetically engineered thermophilic streptococcus with the epsE gene knocked out is used to prepare sugar-free / low-sugar dairy products and reduce the sugar content of dairy products.

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