Method for regulating and controlling lactobacillus casei exopolysaccharide biosynthesis based on regulatory factors CcpA and PurR

Abstract

The invention relates to a method for regulating and controlling lactobacillus casei exopolysaccharide biosynthesis based on regulatory factors CcpA and PurR. The CcpA and the PurR are used as regulatory factors for biosynthesis of the extracellular polysaccharide of the lactobacillus casei and are both combined with promoters P2169-2170 and P2189-2190, and the promoters P2169-2170 and P2189-2190 are arranged on an eps gene cluster for synthesizing the extracellular polysaccharide EPS. The method for regulating and controlling the biological synthesis of the lactobacillus casei extracellular polysaccharide comprises the following steps: constructing a recombinant expression vector of an overexpression / knockout ccpA gene and / or a recombinant expression vector of an overexpression / knockout purR gene, transferring the recombinant expression vector into lactobacillus casei, regulating and controlling the biological synthesis of the lactobacillus casei extracellular polysaccharide, and further regulating and controlling the flavor and taste of dairy products. Research results of the application greatly expand the regulation and control mechanism of lactobacillus casei EPS biosynthesis, are beneficial to improving the taste and nutritional value of dairy products, and are beneficial to enhancing food safety and quality control.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for regulating the biosynthesis of extracellular polysaccharides of Lactobacillus casei based on regulatory factors CcpA and PurR. Background Art

[0002] Lactobacillus casei has unique fermentation characteristics and probiotic functions, and has broad application prospects in the food industry and animal husbandry production. [1,2] The secondary metabolites exopolysaccharides (EPS) produced by Lactobacillus casei during the fermentation process not only have an important influence on the texture, rheology and flavor of dairy products, but also have multiple biological activities such as anti-tumor, anti-oxidation, immunomodulation, activation of immune cells and enhancement of the secretion of immune molecules in the body. [3,4] .

[0003] At present, the research on Lactobacillus casei exopolysaccharides mainly focuses on the yield and structure. [5-7] However, there is a lack of reports on the transcriptional regulatory mechanisms of exopolysaccharide biosynthesis.

[0004] [1]DANTAS AB,JESUS ​​VF,SILVA R,et al.Manufacture of probiotic MinasFrescal cheese with Lactobacillus casei Zhang[J].Journal of Dairy Science,2016,99(1):18-30.

[0005] [2]NIGHSWONGER BD, BRASHEARS MM, GILLILAND S E. Viability of Lactobacillus acidophilus and Lactobacillus caseiin fermented milk products during refrigerated storage [J]. Journal of Dairy Science, 1996, 79(2): 212-9.

[0006] [3]LIU C F,TSENG K C,CHIANG S S,et al.Immunomodulatory andantioxidant potential of Lactobacillus exopolysaccharides[J].Journal of theScience of Food and Agriculture,2011,91(12):2284-91.

[0007] [4]LIU C T,CHU F J,CHOU C C,et al.Antiproliferative and anticytotoxiceffects of cell fractions and exopolysaccharides from Lactobacillus casei 01[J].Mutation Research,2011,721(2):157-62.

[0008] [5]LI N,HUANG Y,LIU Z,et al.Regulation of EPS production inLactobacillus casei LC2W through metabolic engineering[J].Letters in AppliedMicrobiology,2015,61(6):555-61.

[0009] [6]LI N,WANG Y,ZHU P,et al.Improvement of exopolysaccharideproduction in Lactobacillus casei LC2W by overexpression of NADH oxidase gene[J].Microbiology Research,2015,171:73-7.

[0010] [7]MINARI G D,PIAZZA R D,SASSD C,et al.EPS production byLacticaseibacillus casei using glycerol,glucose,and molasses as carbonsources[J].Microorganisms,2024,12(6):1159-75. Summary of the invention

[0011] Based on the above problems existing in the prior art, the purpose of the present application is to provide a method for regulating the biosynthesis of exopolysaccharides of Lactobacillus casei based on regulatory factors CcpA and PurR.

[0012] The present invention identifies two transcriptional regulatory factors related to the biosynthesis of exopolysaccharides in Lactobacillus casei, and determines the interaction between them and the promoter on the exopolysaccharide gene cluster, confirming their regulatory effect on the synthesis of exopolysaccharides.

[0013] The purpose of this application can be achieved through the following technical solutions:

[0014] One of the technical solutions of the present application provides the use of the ccpA gene in regulating the biosynthesis of extracellular polysaccharides of Lactobacillus casei, wherein the nucleotide sequence of the ccpA gene is shown in SEQ.ID.No.1, the ccpA gene expresses the CcpA protein, and the amino acid sequence of the CcpA protein is shown in SEQ.ID.No.2; the CcpA protein acts as a regulatory factor and is involved in the promoter region P on the eps gene cluster that synthesizes the extracellular polysaccharide EPS. 2169-2170 and P 2189-2190 In combination, the promoter region P 2169-2170 The nucleotide sequence of the promoter region P is shown in SEQ.ID.No.3. 2189-2190 The nucleotide sequence is shown in SEQ.ID.No.4; the promoter region P 2169-2170 and P 2189-2190 Regulate the expression of eps gene and then regulate the synthesis of Lactobacillus casei exopolysaccharides.

[0015] Furthermore, CcpA (Catabolite control protein A) is a regulatory factor found in many bacteria, which plays an important role in the regulation of carbon source metabolism. The functions of CcpA mainly include the following aspects: (1) Carbon source priority regulation: CcpA participates in the preferential utilization of carbon sources. When there are multiple carbon sources in the environment, CcpA ensures that bacteria first utilize more optimal and energy-efficient carbon sources, such as glucose, and inhibits the metabolic pathways of other secondary carbon sources until the main carbon source is exhausted. (2) Metabolic gene expression regulation: CcpA directly or indirectly regulates the transcription of related genes by binding to specific DNA sequences (called catabolite-responsive elements or CREs). These genes are generally involved in the intake and decomposition of carbohydrates, as well as other metabolic processes. (3) Enzyme activity regulation: CcpA can bind to the precursors (zymogens) of some enzymes to promote their maturation and activation, or inhibit the activity of certain enzymes, thereby affecting metabolic pathways. (4) Energy management: The role of CcpA also extends to the management of energy metabolism, ensuring that bacteria maintain energy efficiency and adaptability under different growth conditions. (5) Stress response: In some cases, CcpA may participate in the bacterial response to environmental changes or stress conditions, such as hypoxia or nutrient limitation, by regulating the expression of related genes to help cells adapt. In short, as a global regulatory factor, CcpA ensures that bacteria can effectively utilize and manage carbon sources and maintain a balance between growth and survival under different environmental conditions by finely regulating gene expression and enzyme activity.

[0016] The second technical solution of the present application provides the use of the purR gene in regulating the biosynthesis of extracellular polysaccharides of Lactobacillus casei, wherein the nucleotide sequence of the purR gene is shown in SEQ.ID.No.5, the purR gene expresses the PurR protein, and the amino acid sequence of the PurR protein is shown in SEQ.ID.No.6; the PurR protein acts as a regulatory factor and interacts with the promoter region P on the eps gene cluster that synthesizes the extracellular polysaccharide EPS. 2169-2170 and P 2189-2190 Combined.

[0017] Furthermore, PurR regulatory factors play a key role as transcription factors in microorganisms, especially bacteria, and are mainly responsible for regulating the expression of genes related to purine metabolism. The main functions of PurR regulatory factors are: (1) Regulation of purine metabolism: PurR regulatory factors directly bind to the promoter regions of genes related to purine synthesis and utilization, and in this way control the transcription of these genes. When the level of purine nucleotides (such as adenosine triphosphate ATP and guanosine triphosphate GTP) in the cell is sufficient, PurR binds to these nucleotides, and its conformational changes cause it to dissociate from the promoter region, allowing gene transcription to proceed, thereby maintaining the balance of purine metabolism. (2) Energy conservation: In the case of high purine content, inhibiting the expression of purine synthesis genes helps to save energy and reduce the accumulation of metabolic intermediates, which is an adaptive strategy of the cell. (3) Metabolic homeostasis: PurR, as a negative regulatory factor, helps cells maintain metabolic homeostasis and prevent excessive synthesis of purines, which is essential for cell growth and survival. (4) Responding to environmental changes: When the environment changes or nutrients are limited, if the intracellular purine level decreases, the ligand binding state of PurR will change, causing it to dissociate from the promoter, thereby activating the expression of purine synthesis genes and ensuring that the cell can adapt to new environmental conditions. (5) Global impact: In addition to purine metabolism, PurR regulators may also affect other biological processes because they may have cross-regulation with other metabolic pathways or physiological processes, depending on the genome and physiological needs of specific microorganisms. In summary, PurR regulators are key regulators of purine metabolism in microbial cells. They dynamically adjust the expression of related genes by sensing the intracellular purine level, thereby ensuring the efficiency and adaptability of cellular metabolism.

[0018] Furthermore, there are many promoter elements on the eps gene, such as P 2169-2170 , P 2175-2176 ,P 2180-2181 ,P 2183-2184 ,P 2185-2186 ,P 2189-2190 These promoter elements play a key role in the regulation of gene expression. They are part of the DNA sequence, located upstream of the gene, and are responsible for initiating the transcription process. Promoter elements ensure that the correct gene is transcribed at a specific time and under specific circumstances by binding to RNA polymerase and transcription factors.

[0019] Furthermore, the present application verified through electrophoresis assay (EMSA) that both the CcpA regulatory factor and the PurR regulatory factor can bind to the promoter element P 2169-2170 and P 2189-2190 The promoter P 2169-2170It is the promoter of an operon, controlling the expression of five genes: LC2W-2170, LC2W-2171, LC2W-2172, LC2W-2173 and LC2W-2174. 2189-2190 It is the promoter of an operon, controlling the expression of the four genes LC2W-2186, LC2W-2187, LC2W-2188 and LC2W-2189. The genes LC2W-2170, LC2W-2171, LC2W-2172, LC2W-2173, LC2W-2174, LC2W-2186, LC2W-2187, LC2W-2188 and LC2W-2189 and the promoters are all on the eps gene cluster. For details, please refer to the inventor's previous article Song Xin, et al. Relationship Between Putative eps Genes and Production of Exopolysaccharide in Lactobacillus casei LC2W. Frontiers in microbiology 9. (2018): 1882.

[0020] In some specific embodiments, in order to study the promoter P 2169 -2170 and P 2189-2190 Precise binding site, promoter P 2169 -2170 It is divided into three parts: M1 (1-100 bp), M2 (56-154 bp) and M3 (100-198 bp). 2189-2190 It is divided into three parts: M4 (1-120 bp), M5 (121-246 bp) and M6 (247-359 bp). The CcpA regulatory factor binds to the promoter P with different affinities. 2169-2170 The M1, M2, M3 sites and promoter P 2189-2190 The PurR regulatory factor binds to the promoter P 2169-2170 The M3 site and P 2189-2190 The M6 ​​site is used to regulate eps gene expression.

[0021] A third technical solution of the present invention provides a method for regulating the biosynthesis of Lactobacillus casei exopolysaccharide, the method comprising the following steps:

[0022] A recombinant expression vector for overexpressing / knocking out the ccpA gene and / or a recombinant expression vector for overexpressing / knocking out the purR gene is constructed, and the recombinant expression vector is transferred into Lactobacillus casei, thereby regulating the biosynthesis of Lactobacillus casei exopolysaccharide.

[0023] Furthermore, the Lactobacillus casei is preferably a LC2W strain; the Lactobacillus casei may also be replaced by other types of lactobacillus such as Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus salivarius.

[0024] Furthermore, overexpression of the ccpA gene and knockout of the purR gene have a synergistic effect, which reduces the production of exopolysaccharides of Lactobacillus casei.

[0025] Furthermore, knocking out the ccpA gene and overexpressing the purR gene have a synergistic effect, which increases the production of extracellular polysaccharides of Lactobacillus casei.

[0026] Furthermore, in some specific embodiments of the present invention, overexpression of the ccpA gene resulted in reduced expression levels of LC2W-2170, LC2W-2171, and LC2W-2172, and EPS production was 122.22 mg / mL, a decrease of 21.2%.

[0027] Further, in some embodiments of the present invention, knocking out the purR gene resulted in a 20.2% reduction in EPS production to 123.77 mg / mL. The transcription levels of LC2W-2187, LC2W-2188, and LC2W-2189 decreased by 0.69, 0.70, and 0.64 times, respectively.

[0028] Furthermore, the knockout of ccpA gene or the knockout of purR gene is performed by CRISPR-Cas9 gene editing system. CRISPR-Cas9 gene editing system is a high-efficiency and high-precision gene modification tool. It uses CRISPR RNA (crRNA) and transcription activator-like effector nuclease (Cas9 enzyme) to recognize and cut specific DNA sequences, and then the cell's DNA repair mechanism (such as non-homologous end joining or homology-directed repair) may lead to the knockout of the target gene.

[0029] A fourth technical solution of the present invention provides the use of Lactobacillus casei overexpressing ccpA gene and / or purR gene in regulating the production of extracellular polysaccharides, wherein the Lactobacillus casei overexpressing ccpA gene and / or purR gene contains a ccpA overexpression plasmid and / or a purR plasmid.

[0030] A fifth technical solution of the present invention provides an application of Lactobacillus casei with ccpA gene and / or purR gene knocked out in regulating the production of extracellular polysaccharides, wherein the Lactobacillus casei with ccpA gene and / or purR gene knocked out is cloned by CRISPR-Cas9 D10A The gene knockout was achieved by the gene editing system, which were denoted as LC2W△ccpA and LC2W△purR, respectively.

[0031] In the above application, Lactobacillus casei is used as a probiotic added to dairy products to regulate the sugar content in dairy products and improve the flavor of dairy products.

[0032] A sixth technical solution of the present invention provides an overexpression recombinant vector, wherein the overexpression recombinant vector is used to overexpress the ccpA gene or the purR gene.

[0033] Compared with the prior art, the beneficial effects of the present application are at least in the following aspects:

[0034] (1) The regulatory factors CcpA and PurR play important roles in carbohydrate metabolism and purine biosynthesis, respectively. However, there is no evidence of their connection with EPS biosynthesis in Lactobacillus casei. Using DNA affinity chromatography pull-down and proteomics, two transcriptional regulatory factors CcpA and PurR involved in EPS biosynthesis were identified in Lactobacillus casei.

[0035] (2) The research results of this application have greatly expanded the regulatory mechanism of EPS biosynthesis in Lactobacillus casei. Revealing the regulatory mechanism of EPS synthesis will help deepen the understanding of microbial metabolic networks, provide theoretical references for the study of polysaccharide synthesis in other microorganisms, and promote the development of microbiology and biotechnology.

[0036] (3) EPS has a direct impact on the taste, stability, and shelf life of dairy products. Understanding the regulatory mechanism will help develop lactic acid bacteria products with higher EPS content and improve the taste and nutritional value of dairy products.

[0037] (4) Development of new probiotic products: Through genetic engineering and based on the understanding of the regulatory mechanism, Lactobacillus casei can be modified in a targeted manner to develop products with new probiotic functions, such as improving intestinal microecology and enhancing immunity.

[0038] (5) Enhance food safety and quality control: Understanding the EPS regulation mechanism of Lactobacillus casei can help control the production process more accurately and ensure the consistency and stability of product quality. It can also help prevent possible microbial contamination problems and improve food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 For identification and purification of transcriptional regulatory factors;

[0040] Figure 1 The middle mark indicates: A: promoter P 2189-2190SDS-PAGE of binding proteins; B: LC-MS / MS identification results; C: Expression and purification of CcpA (M: Marker; a: bacterial solution; b: protein supernatant; c: purified protein); D: Expression and purification of PurR (M: Marker; a: bacterial solution; b: protein supernatant; c: purified protein);

[0041] Figure 2 is the ability of CcpA to bind to the promoter on the eps gene cluster;

[0042] Figure 2 The middle mark indicates: A: CcpA and promoter P 2169-2170 B: Interaction between CcpA and promoter P 2189-2190 Interaction between CcpA and promoter P 2180-2181 , P 2183-2184 , P 2185-2186 and P 2187-2188 interactions;

[0043] Figure 3 is the ability of PurR to bind to the promoter on the eps gene cluster;

[0044] Figure 3 The middle mark indicates: A: PurR and promoter P 2169-2170 Interaction between PurR and promoter P 2189-2190 Interaction between PurR and promoter P 2180-2181 , P 2183-2184 , P 2185-2186 and P 2187-2188 interactions;

[0045] Figure 4 CcpA and PurR are linked to the promoter P 2169-2170 and P 2189-2190 The binding site of

[0046] Figure 4 The middle mark indicates: A: CcpA and promoter P 2169-2170 Binding site analysis of CcpA and promoter P 2189-2190 Binding site analysis of PurR and promoter P 2169-2170 Binding site analysis of PurR and promoter P 2189-2190 Analysis of binding sites;

[0047] Figure 5 For the construction of ccpA and purR knockout strains;

[0048] Figure 5Markers in the middle indicate: A: Schematic diagram of gene knockout; B: Verification results of knockout gene ccpA (M: Marker; a: wild-type strain; b and c: knockout strains); C: Verification results of knockout gene purR (M: Marker; a: wild-type strain; b and c: knockout strains)

[0049] Figure 6 The effect of CcpA and PurR on the production of extracellular polysaccharides;

[0050] Figure 6 The marks in the middle indicate: A: the effect of CcpA on the production of extracellular polysaccharides; B: the effect of PurR on the production of extracellular polysaccharides; C: the effect of ccpA on the expression level of genes on the eps gene cluster; D: the effect of purR on the expression level of genes on the eps gene cluster. DETAILED DESCRIPTION

[0051] 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 in conjunction with 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 noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0052] All raw materials of the present invention are not particularly limited in their sources unless otherwise specified, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0053] (I) The materials involved in this application are as follows:

[0054] MRS medium: tryptone 10g / L, yeast extract powder 5g / L, beef extract powder 10g / L, glucose 20g / L, ammonium citrate 2g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1mL / L; solid medium plus agar powder 20g / L.

[0055] LB medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, used to culture E. coli. Add 20 g / L agar powder to the solid medium.

[0056] SMRS medium: MRS medium containing 10% (w / v) glycine.

[0057] MMRS recovery medium: contains 0.5M sucrose, CaCl 2 2 mM, MgCl 2 20 mM MRS medium.

[0058] PBS buffer solution (10×): Na 2 HPO 4 80mM, NaCl 1.36M, KH 2 PO 4 20mM, KCl 26mM, pH 7.4.

[0059] NPI-10: NaH 2 PO 4 ·H 2 O 50 mM, NaCl 300 mM, imidazole 10 mM, pH 8.0.

[0060] NPI-20: NaH 2 PO 4 ·H 2 O 50 mM, NaCl 300 mM, imidazole 20 mM, pH 8.0.

[0061] NPI-250: NaH 2 PO 4 ·H 2 O 50 mM, NaCl 300 mM, imidazole 10 mM, pH 8.0.

[0062] (II) The instruments and equipment involved in this application are as follows:

[0063] Plate Smart small plate centrifuge, Spectra Max i3x multifunctional microplate reader, fully automatic growth curve analyzer, Nanodrop 2000 ultra-micro nucleic acid protein quantification instrument, S1000 PCR instrument, Power basic electrophoresis instrument, ChemiDoc XRS+ gel imager, Micropulse electroporation transformation instrument.

[0064] Example 1 Preparation and transformation of competent cells of Escherichia coli and Lactobacillus casei

[0065] Preparation of competent E. coli: Streak the E. coli Top10 strain (purchased from Shanghai Bohu Biotechnology Co., Ltd., product number BH-J011050) stored in -80℃ glycerol on an LB plate and culture at 37℃ for 12-24h until the colony diameter reaches 1-2mm. Pick a single colony in a test tube containing 4mL LB medium and culture on a shaker for 12h (37℃, 200rpm). Inoculate 50mL LB medium at a 1% inoculum and culture on a shaker until OD 600 = 0.3-0.5, centrifuge (4°C, 4500 rpm, 10 min) to collect the cells. 2The solution was gently washed twice and then 1 mL of 0.1 mol / L CaCl containing 15% glycerol was added. 2 The resuspended bacteria were dispensed into 1.5 mL sterilized EP tubes at 100 μL per tube and stored in a -80°C refrigerator for later use.

[0066] E. coli transformation experiment: After the competent cells are melted on ice, 10 μL of the ligation product is added and gently mixed, and ice bath is placed for 30 minutes. After heat shock at 42°C for 90 seconds, it is placed on ice for 3 minutes, and 900 μL of fresh LB liquid medium is added. Incubate on a shaking table for 45-60 minutes (37°C, 200 rpm). Discard 900 μL of supernatant by centrifugation (4500 rpm, 5 minutes), resuspend the remaining 100 μL of bacterial solution and spread it on the resistance plate, and culture at 37°C for 24-48 hours. Pick the clones on the resistance plate and add 10 μL of sterile water. After pipetting and mixing, take 0.5 μL of bacterial solution as a template, use the designed verification primers for PCR amplification, and detect the size of PCR products by gel electrophoresis.

[0067] Preparation of competent state of Lactobacillus casei: streak the glycerol-preserved Lactobacillus casei LC2W strain (purchased from China Microorganism Collection Center, strain collection number CGMCC NO.0828) on MRS solid plate and culture at 37°C until the diameter of single colony reaches 1-2mm (streaking two generations). Pick a single colony and inoculate it in 15mL MRS medium and let it stand at 37°C for 12h. Inoculate the activated bacterial solution in 50mL SMRS medium and let it stand at 37°C until OD 600 =0.5~0.6. Collect the cells by centrifugation (4℃, 4000g, 10min) and add 10% glycerol solution to wash the precipitate. Resuspend the cells with 1mL pre-cooled 10% glycerol solution, dispense into 1.5mL sterilized EP tubes at 90μL per tube, and store in a -80℃ refrigerator for later use.

[0068] Lactobacillus casei electrotransformation experiment: Add knockout plasmid or overexpression plasmid to competent cells, mix thoroughly and transfer to a 2mm electrode cup pre-cooled on ice. After electric shock (voltage is 2kv), quickly add 900μL recovery medium MMRS. Transfer the bacterial solution in the electrotransformation cup to a 1.5mL sterilized EP tube and let it stand at 37℃ for 2h. After centrifugation, discard 900μL of supernatant, resuspend the remaining 100μL of bacteria, apply it to the resistance plate, and invert it anaerobically at 37℃ for 48h.

[0069] Example 2 Plasmid Construction

[0070] This example involves PCR and seamless cloning, and the operation is as follows:

[0071] (1) PCR amplification reaction system and program settings

[0072] 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 ddH 2 O. PCR amplification program: 95℃3min; 98℃10s; Tm (primer annealing temperature) 30s; 72℃30s / kb, 30 cycles; 72℃10min. 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 recovered DNA concentration was determined using the ultra-micro nucleic acid protein quantifier Nanodrop 2000.

[0073] (2) Seamless cloning reaction system

[0074] The target fragment amplified by PCR and the linearized vector were recovered by gel recovery kit, and the concentration of the recovered DNA was determined by ultra-micro nucleic acid protein quantification instrument Nanodrop 2000. The ligation was completed according to the steps of Clon Express II one-step cloning kit, the molar ratio of vector and target fragment was 1:2, and the 10μL reaction system included 0.4pmol of target fragment, 0.2pmol of vector fragment, 2μL of 5×CEⅡBuffer, 1μL of Exnase and ddH 2 O.

[0075] Based on the above technologies, knockout plasmids, overexpression plasmids, and protein purification plasmids were constructed:

[0076] Construction of knockout plasmid: Using the genome of Lactobacillus casei LC2W as a template (the target gene fragment on the genome contains the upstream and downstream homologous arms of the gene ccpA, CcpA-up and CcpA-down, the sequence is shown in SEQ.ID.No.1), CcpA-up-F / R and CcpA-down-F / R as primers, amplify the upstream and downstream homologous arms of the gene ccpA, CcpA-up and CcpA-down. r ,P 23 -Cas9 D10A , the sequence is shown in SEQ.ID.No.7, P ldh -sgRNA, sequence as shown in SEQ.ID.No.8, Has(LC2W_2179), repD, repE, and Em rgene from pIB184) as a template, using primers sgRNA-CcpA-F / R, amplify the CcpA-sgRNA fragment. Using CcpA-up, CcpA-down and CcpA-sgRNA as templates, CcpA-up-F / sgRNA-CcpA-R as primers, overlap PCR amplification obtained the CcpA-DNA fragment. The CcpA-DNA fragment and pLCNICK (Xba I / Apa I) were seamlessly cloned and connected, and the knockout plasmid LC2W△ccpA was obtained by kanamycin resistance screening. The construction method of the gene purR knockout plasmid LC2W△purR is similar.

[0077] Overexpression plasmid construction: Using the Lactobacillus casei LC2W genome as a template (the target gene fragment on the genome includes genes ccpA and purR, and the sequences are shown in SEQ.ID.No.1 and SEQ.ID.No.5), CcpA-PIB-F / R and PurR-PIB-F / R are used as primers to amplify genes ccpA and purR. pIB184 was digested with restriction endonucleases EcoR I and BamH I to obtain a linear vector. The target gene and the linear vector pIB184 were seamlessly cloned and connected, and the overexpression plasmids LC2W-ccpA and LC2W-purR were screened for erythromycin resistance.

[0078] Construction of protein purification plasmid: Using the genome of Lactobacillus casei LC2W as a template, primers N-his-ccpA-F / R and N-his-purR-F / R were used to amplify the target genes ccpA and purR. After agarose gel electrophoresis verified the correct bands, they were purified and recovered. The target gene and pET30a plasmid were digested with restriction enzymes Nde I and Xho I, respectively, to obtain a linear vector. The target gene and the linearized vector were connected by T4 enzyme and the product was transferred into the competent E. coli Top10. Plasmids pET30a-ccpA and pET30a-purR were selected for kanamycin resistance. .

[0079] The sequences involved above are shown in Table 1 below.

[0080] Table 1 Primer sequences

[0081] CcpA-up-F ACGGATCACATCTTTTTCTAAACTAGGGCCCGGCGAAATCCAAGCACTATT CcpA-up-R TTTTCACGACTAAGGAGAAAATCAATTGTAATGCCAGCTTTTGTGAGC CcpA-down-F GCTCACAAAAGCTGGCATTACAATTGATTTTCTCCTTAGTCGTGAAAA CcpA-down-R GCACCGAGTCGGTGCTTTTTTTGAGGTTAAATTGGCCTCTAAACAGACATCG sgRNA-CcpA-F CTCAAAAAAAGCACCGACT sgRNA-CcpA-R AAGAAAGGATGATATCACCTCTAGAAAAACCTTCTTGCGAGTAGCGTTTTAGAGCTAGA purR-up-F ACGGATCACATCTTTTTCTAAACTAGGGCCCGCGACAAGAGGCCAAAGAAT purR-up-R GTGTTAGGAGCGAAATGATGTGAGTGCTTATCTCGGACCG PurR-down-F CGGTCCGAGATAAGCACTCACATCATTTCGCTCCTAACAC PurR-down-R GCACCGAGTCGGTGCTTTTTTTGAGCGTTAAAAATGATGACCGCA sgRNA-PurR-F CTCAAAAAAAGCACCGACT sgRNA-PurR-R AAGAAAGGATGATATCACCTCTAGAATCTAGAAAATTGCCAGCTGGTTTTAGAGCTAGA CcpA-PIB-F TATGAATGACAATGATGTTGGATCCTTGGAAAAGCAAACAATTAC CcpA-PIB-R CGATAGATCTCGAGCTCTAGAATTCTTATTTCGTTGAACCACGCT PurR-PIB-F CGGGATCCATGAAAGTAAGACGAAGTGACCGG PurR-PIB-R CGAGCTCTCATTGACCAGCCACCTCGA N-his-ccpA-F GGAATTCCATATGCACCATCATCATCATCATTTGGAAAAGCAAACAATTAC N-his-ccpA-R CCGCTCGAGTTATTTCGTTGAACCACGCT N-his-purR-F GGAATTCCATATGCACCATCATCATCATCATATGAAAGTAAGACGAAGTGACCGG N-his-purR-R CGGGATCCCACCATCATCATCATCATTCATTGACCAGCCACCTCGA

[0082] Example 3 Proteomics determination of Lactobacillus casei EPS transcription factors

[0083] To identify transcription factors in Lactobacillus casei LC2W and explore their mechanism of action, DNA affinity chromatography was used to isolate potential regulatory proteins. 2189-2190The bound proteins were isolated by coupling to streptavidin magnetic beads and identified by LC-MS / MS.

[0084] The protein expression and purification steps involved in this example are as follows:

[0085] Plasmids pET30a-ccpA and pET30a-purR were transferred into competent E. coli BL21 (DE3) respectively. After kanamycin resistance screening, the correct single colony was picked and activated in 4 mL of LB liquid medium containing kanamycin resistance for 12 hours. 3% was inoculated into 100 mL of LB liquid medium containing kanamycin resistance and cultured in a shaking incubator at 37°C until OD 550 About 0.6, add IPTG at a final concentration of 0.8mmol / L and kanamycin at a final concentration of 50μg / mL, and continue to culture for 8h. Collect the bacteria by centrifugation (4℃, 8000rpm, 10min), wash the bacterial precipitate twice with 25mL pre-cooled PBS buffer, take 20mL pre-cooled PBS buffer to resuspend the bacteria, and crush the resuspended liquid on ice with an ultrasonic crusher. The procedure is as follows: working power 800W, work 5s, pause 5s, and work time is 5min. After crushing, separate the precipitate and supernatant by centrifugation (4℃, 8000rpm, 10min). Take 20μL of the original bacterial solution, ultrasonically crush the supernatant and precipitate (PBS resuspension), add loading buffer for treatment, and observe protein expression with 12.5% ​​SDS-PAGE.

[0086] Take appropriate amount of Ni 2+ -NTA agarose was filled into the affinity chromatography column, and 10 mL of NPI-10 buffer was added to balance Ni 2+ When the liquid in the column is not drained, the supernatant after crushing is gently loaded into the chromatography column through a 0.45μm needle filter. 2+ Add 40 mL of NPI-20 to the column to fully elute the impurities. 2+ 10 mL of NPI-250 was added to the column to elute and collect the target protein. The eluted target protein was transferred to an ultrafiltration tube and concentrated by centrifugation (4°C, 5000 g, 20-30 min).

[0087] The experimental results are as follows Figure 1 shown.

[0088] Promoter P 2189-2190 SDS-PAGE of bound proteins ( Figure 1A) shows that the preliminary binding results showed 6 significantly enriched protein bands, distributed between 10-15kDa, 20-25kDa, 25-35kDa and 50-70kDa (the yellow box circled part). Combining the coverage and protein molecular weight, the carbon metabolism regulatory protein CcpA and the pur operon repressor protein PurR were screened from the protein library as potential transcriptional regulatory factors for EPS biosynthesis ( Figure 1 B). Bioinformatics prediction indicated that they were expressed in the supernatant as soluble proteins. To confirm the interaction between CcpA and PurR and the promoter associated with the eps operon, soluble proteins of CcpA and PurR were labeled with 6×His-tag and purified in Escherichia coli BL21(DE3) ( Figure 1 C and D).

[0089] The DNA affinity chromatography technique, streptavidin magnetic bead coupling, SDS-PAGE, and 6×His-tag labeling described in this example are all routine operations performed by those skilled in the art and can be performed according to conventional methods, so no further details will be given here.

[0090] The conditions of the LC-MS / MS are:

[0091] Data acquisition software: Thermo Xcalibur 4.0 (Thermo, USA)

[0092] Reverse phase column information: C18 column (75μm×25cm, Thermo, USA)

[0093] Chromatographic instrument: EASY-nLC 1200 (Thermo, USA)

[0094] Mass spectrometer: Q_Exactive HF-X (Thermo, USA)

[0095] Chromatographic separation time: 60min

[0096] A: 2% acetonitrile 0.1% formic acid

[0097] B: 80% acetonitrile 0.1% formic acid

[0098] Flow rate: 300nL / min

[0099] gradient:

[0100]

[0101] MS scan range (m / z): 300-1500, acquisition mode: DDA;

[0102] Top 20 (select the 20 most powerful precursor ions for secondary fragmentation);

[0103] Primary mass spectrometer resolution: 60000, AGC target: 3e 6 , Maximum injection time: 20ms, fragmentation method HCD;

[0104] Secondary resolution: 15000, AGC target: 1e 5 , maximum injection time: 50ms, fixed first mass: 100m / z; minimum AGC target: 8e 3 , Intensity threshold: 1.6e 5 , dynamic exclusion time: 18s.

[0105] Example 4 CcpA and PurR bind to promoters in the eps gene cluster

[0106] The global regulatory factor CcpA binds to multiple cre sites in the eps gene cluster, thereby regulating the biosynthesis of exopolysaccharides in plant lactococcus, lactococcus casei and streptococcus pneumoniae. PurR participates in multiple physiological activities such as purine synthesis, pyrimidine synthesis and glycine metabolism. However, there is no data showing the connection between the biosynthesis of exopolysaccharides in Lactobacillus casei and the transcriptional regulatory factors CcpA and PurR. Therefore, the application utilizes EMSA to analyze the interaction between the promoter and CcpA and PurR in the eps gene cluster.

[0107] The operation of the EMSA (gel shift assay) is as follows:

[0108] EMSA detection used 4% native polyacrylamide gel (Native-PAGE), gel system: 500μL 5×TBE Buffer, 500μL 50% glycerol, 1mL 30% Acr-Bis (29:1), 3mL ddH 2 0, 30μL 10% APS, 2.5μL TEMED. Electrophoresis parameters: voltage 170V (10V / cm), time 40min, electrophoresis buffer 0.5×TBE, temperature 4°C. The protein to be analyzed was incubated with the biotinylated promoter at room temperature (20°C) for 20min, then 1μL EMSA / Gel-Shift loading buffer (colorless, 10×) was added and mixed, and the blank group was marked with EMSA / Gel-Shift loading buffer (blue, 10×).

[0109] Transfer parameters: current 380mA, time 60min, transfer buffer 0.5×TBE, temperature 4°C. UV crosslinking parameters: time 60s, working wavelength 254nm, power 1200mJ (120mJ / cm 2). The blocking, labeling, washing and color development were completed according to the instructions of the chemiluminescence EMSA kit, and the gel imager was used for chemiluminescence color development and imaging.

[0110] The experimental results are as follows Figure 2 and Figure 3 shown.

[0111] like Figure 2 A and Figure 2 As shown in B, with the increase of CcpA concentration, the biotinylated promoter P 2169-2170 and P 2189-2190 The migration of the band was blocked, indicating that CcpA was bound to the promoter P 2169-2170 and P 2189-2190 In contrast, the promoter P 2180-2181 , P 2183-2184 , P 2185-2186 and P 2187-2188 No significant band migration was observed, indicating that there was no interaction between them and CcpA ( Figure 2 C) Similarly, PurR also binds to the promoter P 2169-2170 and P 2189-2190 Binds to the promoter P 2180-2181 , P 2183-2184 , P 2185-2186 and P 2187-2188 Combined with Figure 3 ).

[0112] Example 5 Identification of the binding sites of CcpA and PurR to promoters

[0113] In order to further explore the relationship between CcpA and PurR and promoter P 2169-2170 and P 2189-2190 Precise binding site, promoter P 2169 -2170 It is divided into three parts: M1 (1-100 bp), M2 (56-154 bp) and M3 (100-198 bp). 2189-2190 It is divided into three parts: M4 (1-120 bp), M5 (121-246 bp) and M6 (247-359 bp). 2169-2170 or P 2189-2190 The promoter probe was used for EMSA detection.

[0114] The EMSA detection is the same as the operation in the above embodiment. Figure 4 shown.

[0115] like Figure 4 As shown, after incubation with CcpA, M1, M2 and M3 all showed obvious band shift ( Figure 4A) The migration of M1 band is the most obvious, indicating that the promoter P 2169-2170 All three parts of the promoter have the function of binding to CcpA, among which the binding site of M1 is the strongest. Considering that there is no overlap between the M1 and M3 regions, it is reasonable to assume that 2169-2170 There are at least two binding sites in the LC2W-2169 gene. Since the transcription directions of genes LC2W-2169 and LC2W-2170 are different (refer to the inventor's paper: Identification of the promoter of the exopolysaccharide biosynthesis gene cluster of Lactobacillus casei LC2W. Industrial Microbiology 50.4 (2020): 5.), it is speculated that CcpA binds to P 2169-2170 Different sites of P, thereby simultaneously controlling the transcription of genes LC2W-2169 and LC2W-2170, thereby affecting the expression level of the eps gene cluster. 2189-2190 In the promoter, M4 and M6 showed obvious band migration due to the addition of CcpA, and the binding ability of M4 was stronger than that of M6, indicating that CcpA also bound to P 2189-2190 The M4 and M6 regions of the promoter ( Figure 4 B). Based on gene function and transcription direction, it is speculated that CcpA binds to M4 rather than M6 to regulate the transcription of the eps gene.

[0116] PurR and promoter P 2169-2170 and P 2189-2190 EMSA experiments were performed on the three regions of the 100 μg / ml protein, and the results showed that only the M3 probe showed obvious band migration after incubation with PurR ( Figure 4 C), indicating that the promoter P 2169-2170 There is a PurR binding site in the first 98 bp of the promoter. 2189-2190 , the binding phenomenon only occurred in the M6 ​​region, indicating that there is a PurR binding site 120 bp upstream of the gene LC2W-2190 ( Figure 4 D). Combined with RT-qPCR experiment ( Figure 6 D), PurR and promoter P 2189-2190 The M6 ​​of promoter P regulates the gene expression levels of LC2W-2187, LC2W-2188 and LC2W-2189. 2169-2170 M3 and P 2189-2190 M6 has a binding site for PurR.

[0117] Example 6 Effects of CcpA and PurR on Lactobacillus casei EPS

[0118] (1) Construction of ccpA and purR overexpressing strains LC2W-ccpA and LC2W-purR

[0119] To investigate how CcpA and PurR regulate the expression of 2169-2170 and P2189-2190 To affect the biosynthesis of extracellular polysaccharides, CcpA and PurR were first overexpressed and LC2W-ccpA and LC2W-purR overexpression strains were obtained. The EPS production and the transcription level of the eps operon were determined using the wild-type LC2W strain as a control.

[0120] (2) Construction of ccpA and purR knockout strains LC2W△ccpA and LC2W△purR

[0121] This embodiment is also based on CRISPR-Cas9 D10A The ccpA and purR knockout strains LC2W△ccpA and LC2W△purR ( Figure 5 ). The CRISPR-Cas9 D10A Gene editing operations are routine operations for those skilled in the art and can be performed according to conventional methods, so no further elaboration is required here.

[0122] The method for measuring the EPS (exopolysaccharide) yield is as follows:

[0123] Lactobacillus casei LC2W was inoculated into MRS medium and cultured at 37°C for 12h as seed liquid. The seed liquid was inoculated into 30mL MRS medium with an inoculum of 3% (v / v) and cultured at 37°C for 24h. The supernatant was separated by centrifugation (4°C, 8000g, 10min) and 80% (w / v) trichloroacetic acid was added to a final concentration of 4% (w / v). After standing at 4°C for 8h, the impurities in the precipitate were removed by centrifugation (4°C, 8000g, 10min). The supernatant was transferred to a dialysis bag (cutoff 8000-14000Da) for dialysis, and the water was changed every 4h for a total of 72h. The galactose standard curve was prepared by the phenol-sulfuric acid method and the exopolysaccharide yield was determined.

[0124] The transcription level of the eps operon is determined by real-time quantitative PCR (RT-qPCR), comprising the following steps:

[0125] (1) RNA extraction

[0126] Pick a single colony of engineered bacteria and culture it to the stable phase. After centrifugation to collect the bacterial precipitate, add 200 μL lysozyme (20 mg / mL) and react at 37°C for 20 minutes. Add 1 mL of Trizol solution to each tube and shake vigorously for 30 seconds, then let it stand on ice for 5 minutes. Obtain the supernatant by centrifugation (4°C, 12000g, 10 minutes) and add 200 μL chloroform (0.2 volume of Trizol) to 500 μL of supernatant. Mix thoroughly and let it stand on ice for 15 minutes. Separate the upper aqueous phase by centrifugation (4°C, 12000g, 15 minutes) and add 700 μL isopropanol, gently invert and mix, and let it stand on ice for 10 minutes. Precipitate the RNA at the bottom of the tube by centrifugation (4°C, 12000g, 10 minutes) and add 1.0 mL 75% ethanol (prepared with DEPC water, pre-cooled), gently invert and mix, and wash the precipitate. Repeat the washing and precipitation step once, discard the supernatant and dry at room temperature for 5 minutes. Resuspend the RNA in each tube with 25-30 μL DEPC water and store at -80°C.

[0127] (2) Real-time quantitative PCR (RT-qPCR)

[0128] The genomic DNA in RNA was removed and reverse transcribed into cDNA according to the PrimeScriptRT kit, which was used as the template for RT-qPCR. The reaction system included 0.5 μL cDNA, 0.5 μL upstream and downstream primers, 10 μL SYBR Premix Ex Taq and 8.5 μL ddH 2 O. RT-qPCR reaction conditions: pre-denaturation 95℃30s, 95℃5s, 60℃30s, 40 cycles. With recA as the internal reference gene, according to formula 2 -△△Ct Calculate the target gene expression level.

[0129] The experimental results are as follows Figure 6 shown.

[0130] Overexpression of ccpA gene resulted in a 17.7% decrease in exopolysaccharide production, whereas ccpA deficiency resulted in a 5.7% increase in exopolysaccharide production ( Figure 6 A). To determine whether CcpA affects the expression level of eps, RT-qPCR was used for detection. Compared with the wild-type strain, ccpA deficiency resulted in a significant increase in the expression levels of genes LC2W-2170, LC2W-2171, and LC2W-2172 by 3.81-fold, 2.43-fold, and 2.51-fold ( Figure 6C). In addition, other genes in the eps operon did not show significant changes in transcriptional levels. The transcriptional levels of genes LC2W-2171 and LC2W-2172 in the ccpA overexpression strain decreased by 0.5 and 0.6 times, respectively. In summary, CcpA acts as a negative transcriptional regulator, inhibiting the expression levels of genes LC2W-2171 and LC2W-2172, thereby inhibiting the production of exopolysaccharides.

[0131] Compared with CcpA, knockout of purR resulted in an 18.0% decrease in exopolysaccharide production, whereas overexpression of purR resulted in a 5.6% increase in exopolysaccharide production ( Figure 6 B). RT-qPCR results showed that the expression levels of genes LC2W-2187, LC2W-2188, and LC2W-2189 in the purR knockout strain increased by 2.22, 2.34, and 3.15 times, respectively. The transcription levels of genes LC2W-2187, LC2W-2188, and LC2W-2189 in the purR overexpression strain decreased by 0.69, 0.70, and 0.6 times, respectively. Figure 6 D). In summary, PurR, as a transcriptional activator, positively regulates the transcription levels of genes LC2W-2187, LC2W-2188, and LC2W-2189, thereby promoting the biosynthesis of EPS.

[0132] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that 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 work. Therefore, the present invention is not limited to the above embodiments, and 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.

[0133] The sequences involved in this application are:

[0134] SEQ.ID.No.1 (CcpA nucleotide sequence information):

[0135] TTGGAAAAGCAAACAATTACAATTTATGATGTCGCACGCGAGGCTAATGTCT

[0136] CAATGGCGACAGTGTCTCGAGTCGTTAATGGTAACCCAAATGTGAAACCAG

[0137] CTACTCGCAAGAAGGTTTTGGAAGTGATCGAACGGCTTGATTATCGGCCGA

[0138] ATGCAGTTGCTCGTGGTCTGGCAAGCAAGAAGACGACAACTGTCGGGGTAA

[0139] TTATCCCAGATGTCACGAACATGTTCTTCTCAAGCTTGGCACGTGGGATTGA

[0140] TGATGTTGCCACGATGTACAAGTACAATATCATCTTGGCGAACTCTGATGAA

[0141] AACAATCAAAAAGAAGTCACGGTGTTAAACACATTGTTGGCTAAACAAGTG

[0142] GATGGTTTGATTTTCATGGGCCATGAATTGACTGACTCAATTCGTGCTGAAT

[0143] TCTCACGCAGTAAGACCCCTGTTGTGCTTGCTGGCTCAATCGATCCTGACGA

[0144] ACAGGTTGGCAGTGTCAACATCGATTACGTGGCTGCTGTCGAAGAAGCCAC

[0145] GCGTCAGTTATTGGAAAGCGGCAACAAGCGGGTTGCTCTGGCGACCGGTTC

[0146] ATTGACGCATCCGATCAATGGGCAGTTCCGGCTCAAGGGTTACAAACAGGC

[0147] TCTGGAAAAGGCCGGTGTTGCTTATGACGAAAGCCTGATTTTTGAAAATGAG

[0148] CCAAGCTACCAAGCTGGTTTAGCCTTGTTTGATAAGTTGCAGAAAGTTGGGG

[0149] CAACGGCGGTTATTGCAGGTGATGACGAGTTAGCAGTTGGCCTTTTGGATGG

[0150] CGCAATTGATAAGGGCGTTAAGGTGCCGGACGACTTTGAAATTATCACCAG

[0151] CAATAACACCAAACTGACGGAAATGACTCGGCCACAACTTACTTCGATTGA

[0152] TCAGCCATTGTATGATATTGGTGCGGTTGCTATGCGCTTGCTGACCAAGATG

[0153] ATGAATAAAGAAGAGATCGAGGAAAAGACCGTTATGCTTGGCTTCGATATT

[0154] TTGAAGCGTGGTTCAACGAAATAA

[0155] SEQ.ID.No.2 (CcpA amino acid sequence information):

[0156] MEKQTITIYDVAREANVSMATVSRVVNGNPNVKPATRKKVLEVIERLDYRPNA

[0157] VARGLASKKTTTVGVIIPDVTNMFFSSLARGIDDVATMYKYNIILANSDENNQK

[0158] EVTVLNTLLAKQVDGLIFMGHELTDSIRAEFSRSKTPVVLAGSIDPDEQVGSVNI

[0159] DYVAAVEEATRQLLESGNKRVALATGSLTHPINGQFRLKGYKQALEKAGVAY

[0160] DESLIFENEPSYQAGLALFDKLQKVGATAVIAGDDELAVGLLDGAIDKGVKVPD

[0161] DFEIITSNNTKLTEMTRPQLTSIDQPLYDIGAVAMRLLTKMMNKEEIEEKTVMLGFDILKRGSTK*

[0162] SEQ.ID.No.3 (Promoter P 2169-2170 Nucleotide sequence information):

[0163] ATGACAGTGTACATCAATCATCTTTCAGTTCCCCTTCCAATATGTACTAGTAT

[0164] CTAATGAAGCGCTGACAAGGTCAAATCGTTTTTTGTAACAGTTTGTTAACGA

[0165] TTTGCAATAAAGCCCCATCAGCCGCGCGAGATTCCAAACTTTGCGGTATAAT

[0166] ATTTGAATATTCAGACATTATGGAGGAAATGAGGTTTAGAASEQ.ID.No.4(Promoter P 2189-2190 Nucleotide sequence information):

[0167] AGTTTAGTGAACCCCCAAAAGTTGCATTGCTTATATTTTAAACTAAAAATCA

[0168] GGTTAGTCTGGTTACAAGTTAGGGTCATTCTGACGGGTTTATAGGAAATTGT

[0169] AAAGAGACTGTAATAAAACGTCTTCTTTGGTTAACAACAACGTCAAAAGGG

[0170] GACTCTTTCGACTTTCGACACCAGCAATAGAAGTATTAGAATATGTGAATTA

[0171] ACCAATTATGGGTGGACTTGATTACACAAGCTAGGGTATTATGCTTTGGAGT

[0172] AGCATTATTGAAACTTCTTTTTTAGTTGCGGGCTGCGCTGCTTGCAGTCCGTA

[0173] TTTTAGTTAGTTTCAACAAATCAAGGGATTAGGTGGGGAAAGAGTTASEQ.ID.No.5(PurR nucleotide sequence information):

[0174] ATGAAAGTAAGACGAAGTGACCGGTTAATTGATATGACCCGGTATTTATTA

[0175] GAACGACCGCATACCTTGATTCCACTGACTTTTTTCTCAAAACGGTATGAGA

[0176] GTGCGAAGTCTTCTATCAGTGAAGATCTGGCTATTGTCCGGCGAACATTTGC

[0177] CCAGCGACAGACGGGCATTCTTGAAACGGTGCCAGGCGCAGCAGGCGGTGT

[0178] GCGCTATATTCCGATCATGGGAGAAGATGAGGCGGAAAACTTTATTGCCGC

[0179] AATGTCCAAACGTTTATCCGAGACAGATCGCTTATTGCCAGGCGGCTACGTT

[0180] TATCTTTCCGATCTGTTAGGAACACCTAGTGTGCTACGGCAAATTGGGCGAT

[0181] TAATTGCCACCCAATATCGCGAGGCCCATGTCGATGCTGTCATGACGGTTGC

[0182] AACCAAAGGTATTCCAATTGCGCAAAGTGTTAGTCAGTATCTGAATGTGCCG

[0183] TTTGTGATTGTGCGCCGTGACTCCAAGATTACGGAAGGTTCCACAGTGTCCG

[0184] TGAACTATGTGTCCGGTTCATCTGCGCGAATTGAAAAGATGGAACTGAGTA

[0185] AACGCAGCTTGCCAGCTAACTCAAGGGTTCTGATTGTGGACGACTTCATGAA

[0186] AGGCGGCGGCACCGTCAATGGCATGAAGGCCCTGATTGATGAATTCAATGC

[0187] CAAAATGGTCGGTATCACGGTTTTCGCTGAAGGTAAGTTCGACGGTGATCGG

[0188] ATGGTCAGTGAATATACATCATTGATTCGGGTTGACAAGGTGGACACTAAA

[0189] GCCAATACGCTTCATGCCACAGCTGGCAATTTTCTAGATCAGAATCGTCAAC

[0190] TACTCGAGGTGGCTGGTCAATGA

[0191] SEQ.ID.No.6 (PurR amino acid sequence information):

[0192] MKVRRSDRLIDMTRYLLERPHTLIPLTFFSKRYESAKSSISEDLAIVRRTFAQRQT

[0193] GILETVPGAAGGVRYIPIMGEDEAENFIAAMSKRLSETDRLLPGGYVYLSDLLGT

[0194] PSVLRQIGRLIATQYREAHVDAVMTVATKGIPIAQSVSQYLNVPFVIVRRDSKIT

[0195] EGSTVSVNYVSGSSARIEKMELSKRSLPANSRVLIVDDFMKGGGTVNGMKALID

[0196] EFNAKMVGITVFAEGKFDGDRMVSEYTSLIRVDKVDTKANTLHATAGNFLDQN

[0197] RQLLEVAGQ

[0198] SEQ.ID.No.7 (P in pLCNICK 23 -Cas9 D10Aldh - sgRNA nucleotide sequence information): ACGGATTTTGCCTTTTCCGTCCAGAGCAGTATACCATACTGACGCCTTGCGTCTGTGCGAGTTGACCGTGGGAACTGTTGAATTTCCCACATTCGTGAAAACAGATGCTGGAAACGCTTACGTTATTGGAAGCTTGCTTGAAACAGGATTCACAAGTCTTGCTGTAGTAAGGCTCGACGCCATTTTTTGACAATGGCAAAATCATGAAAAAGTCTATCAAATTTGTTTCAGGGAATTGATAATGTGTTATACTCAACGTGAAATGCAGTTTGCATGCACATAAGAAAGGATGATATCACCTCTAGACAGCATTGGAACAGCGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

Claims

1. The application of ccpA gene in regulating the biosynthesis of exopolysaccharide of Lactobacillus casei, characterized in that: The nucleotide sequence of the ccpA gene is shown in SEQ.ID.No.1, the ccpA gene expresses CcpA protein, and the amino acid sequence of the CcpA protein is shown in SEQ.ID.No.2; the CcpA protein acts as a regulatory factor and interacts with the promoter region P on the eps gene cluster that synthesizes extracellular polysaccharide EPS. 2169-2170 and P 2189-2190 In combination, the promoter region P 2169-2170 The nucleotide sequence of the promoter region P is shown in SEQ.ID.No.

3. 2189-2190 The nucleotide sequence is shown in SEQ.ID.No.4; the promoter region P 2169-2170 and P 2189-2190 Regulate the expression of eps gene and then regulate the synthesis of Lactobacillus casei exopolysaccharides.

2. The application of purR gene in regulating the biosynthesis of exopolysaccharide of Lactobacillus casei, characterized in that: The nucleotide sequence of the purR gene is shown in SEQ.ID.No.5, the purR gene expresses PurR protein, and the amino acid sequence of the PurR protein is shown in SEQ.ID.No.6; the PurR protein acts as a regulatory factor and interacts with the promoter region P on the eps gene cluster that synthesizes extracellular polysaccharide EPS. 2169-2170 and P 2189-2190 Combined.

3. A method for regulating the biosynthesis of Lactobacillus casei exopolysaccharide, characterized in that: The method comprises the following steps: constructing a recombinant expression vector for overexpressing / knocking out the ccpA gene and / or a recombinant expression vector for overexpressing / knocking out the purR gene, transferring the recombinant expression vector into Lactobacillus casei, and further regulating the biosynthesis of Lactobacillus casei extracellular polysaccharides.

4. A method for regulating the biosynthesis of Lactobacillus casei exopolysaccharide according to claim 3, characterized in that, The Lactobacillus casei is a LC2W strain.

5. A method for regulating the biosynthesis of Lactobacillus casei exopolysaccharide according to claim 3, characterized in that, Overexpression of ccpA gene and knockout of purR gene have synergistic effect, which reduces the production of exopolysaccharide of Lactobacillus casei. Overexpression of the ccpA gene resulted in decreased expression levels of LC2W-2170, LC2W-2171, and LC2W-2172; knockout of the purR gene resulted in decreased transcription levels of LC2W-2187, LC2W-2188, and LC2W-2189.

6. A method for regulating the biosynthesis of Lactobacillus casei exopolysaccharide according to claim 3, characterized in that, Knocking out the ccpA gene and overexpressing the purR gene have a synergistic effect, which increases the production of extracellular polysaccharides of Lactobacillus casei.

7. Use of Lactobacillus casei overexpressing ccpA gene and / or purR gene in regulating the production of exopolysaccharides, characterized in that: The Lactobacillus casei overexpressing ccpA gene and / or purR gene contains a ccpA overexpressing plasmid and / or a purR overexpressing plasmid.

8. Use of Lactobacillus casei with ccpA gene and / or purR gene knocked out in regulating the production of extracellular polysaccharides, characterized in that: The ccpA gene and / or purR gene knockout in Lactobacillus casei is synthesized by CRISPR-Cas9 D10A Gene knockout achieved by gene editing system.

9. The use of Lactobacillus casei overexpressing ccpA gene and / or purR gene in regulating the production of exopolysaccharides according to claim 7 or the use of Lactobacillus casei knocking out ccpA gene and / or purR gene in regulating the production of exopolysaccharides according to claim 8, characterized in that: In the application, Lactobacillus casei is used as a probiotic added to dairy products to regulate the sugar content in the dairy products and improve the flavor of the dairy products.

10. An overexpression recombinant vector, characterized in that: The overexpression recombinant vector is used for overexpressing the ccpA gene or the purR gene.

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