Method for preparing bifidus yeast by using Crispr-cas gene editing technology
By constructing a CRISPR activation and interference system in Bifidobacterium longan, the expression of glutamate decarboxylase and succinate semialdehyde dehydrogenase is regulated, and the inefficiency and high cost of GABA production in the prior art is solved, and efficient and safe GABA synthesis is achieved, which is applied to cosmetics, beverages, foods and health care products.
Patent Information
- Application Number
- CN202510423485.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-07-08
AI Technical Summary
The existing methods for producing GABA in biofermentation have problems such as low substrate conversion rate, insufficient process stability, disputes in safety and high cost, and lack stable, efficient and low-cost production technology.
Crispr-cas gene editing technology was used to construct the CRISPR activation system of glutamate decarboxylase and the CRISPR interference system of succinate semialdehyde dehydrogenase, and introduced it into Bifidobacterium longan, regulate its expression, and form a novel dififthyst strain that efficiently synthesizes GABA.
It has achieved efficient synthesis of GABA, reduced production costs, increased yield, enhanced biological activity, improved skin health, and is highly safe. It is suitable for cosmetics, beverages, food and health care products.
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Figure CN120272392A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing Saccharomyces boulardii, in particular to a method for preparing Saccharomyces boulardii using Crispr-cas gene editing technology. Background Art
[0002] As a kind of bacteria, the scientific name of Saccharomyces boulardii is Bifidobacterium, also known as Bifidobacterium, and it is an anaerobic Gram-positive bacillus. The lysate of Saccharomyces boulardii fermentation product is a metabolite, cytoplasmic fragment, cell wall component and polysaccharide complex obtained from the culture, inactivation and decomposition of Bifidobacterium.
[0003] Common Saccharomyces boulardii strains include: Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis. Bifidobacterium longum is one of the most common Saccharomyces boulardii strains, containing multiple subspecies, including Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. suis, Bifidobacterium longum subsp. suillum and Bifidobacterium longum subsp. iuvenis, etc.
[0004] The biosynthesis of GABA is mainly achieved through glutamate decarboxylase (GAD) and succinic semialdehyde dehydrogenase (SSADH). GAD converts glutamate into GABA while consuming CO2. SSADH then catalyzes the conversion of succinic semialdehyde into succinic acid, participating in the key steps of GABA metabolism.
[0005] Currently, the main methods for the biological fermentation production of GABA include: (1) Traditional strain fermentation (such as lactic acid bacteria, Monascus) catalyzes the conversion of L-glutamic acid through glutamate decarboxylase (GAD), with a maximum yield of 19.9 g / L; (2) Co-fermentation of composite strains (yeast + lactic acid bacteria + Rhizopus); (3) Genetic engineering to transform Escherichia coli or yeast to strengthen the metabolic pathway; (4) Plant enrichment method activates endogenous enzymes through germination and hydrothermal treatment.
[0006] However, the above-mentioned biochemically fermented GABA has the following problems: the substrate conversion rate of traditional strain fermentation is low (the utilization rate of L-glutamic acid is only 60%-70%); the process stability of co-fermentation with complex strains is insufficient (the proportion of bacteria in the symbiotic system is difficult to control); the genetic engineering modification method is restricted by regulations and faces safety disputes, and the product is easily hydrolyzed and inactivated; the extraction cost of the plant enrichment method is high (the recovery rate is <80% due to ultrafiltration membrane blockage).
[0007] Therefore, there is currently no stable, efficient, and low-cost method for producing GABA. Summary of the Invention
[0008] The object of the present invention is to provide a method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology. The present invention has the characteristics of obtaining a novel Schizosaccharomyces pombe strain with high-efficiency synthesis of GABA, and the preparation method is simple, efficient, and low-cost.
[0009] The technical solution of the present invention: A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology includes the following steps:
[0010] S1. Select strains and target genes: Use Bifidobacterium longum of Schizosaccharomyces pombe as the basic strain, and use the DNA gene sequences of glutamate decarboxylase and succinic semialdehyde dehydrogenase in the basic strain as the target genes;
[0011] S2. Construct a CRISPR activation system for glutamate decarboxylase to obtain the CRISPRa-VP64 plasmid;
[0012] S3. Construct a CRISPR interference system for succinic semialdehyde dehydrogenase to obtain the CRISPRi-2s plasmid;
[0013] S4. Use the method of electroporation to introduce both the CRISPRa-VP64 plasmid and the CRISPRi-2s plasmid into the basic strain to obtain a novel Schizosaccharomyces pombe strain.
[0014] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, step S2 specifically includes the following steps:
[0015] S2.1 Design the gRNA sequence for activating glutamate decarboxylase: Select a group of gRNA sequences that match the PAM at the 3'-end NGG site of the target sequence in the glutamate decarboxylase gene sequence from the RNA sequence of glutamate decarboxylase, and then synthesize and amplify using PCR to obtain the GAD-gRNA sequence;
[0016] S2.2. Clone the gRNA into the CRISPRa vector: Select pcDNA-dCas9-VP64 as the vector, ligate the pcDNA-dCas9-VP64 vector with the GAD-gRNA sequence to obtain the CRISPRa-VP64 vector;
[0017] S2.3. Extract the CRISPRa-VP64 plasmid: Extract the plasmid from the CRISPRa-VP64 vector to obtain the CRISPRa-VP64 plasmid.
[0018] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, step S3 specifically includes the following steps:
[0019] S3.1. Design the gRNA sequence for inhibiting succinic semialdehyde dehydrogenase: Select a group of gRNA sequences from the RNA sequence of succinic semialdehyde dehydrogenase that match the PAM at the NGG site at the 3' end of the target sequence in the succinic semialdehyde dehydrogenase gene sequence, then synthesize and amplify using PCR to obtain the SSADH-gRNA sequence;
[0020] S3.2. Clone the gRNA into the CRISPRa vector: Select pCBH-CRISPRi-2s as the vector, ligate the pCBH-CRISPRi-2s vector with the SSADH-gRNA sequence to obtain the CRISPRi-2s vector;
[0021] S3.3. Extract the CRISPRi-2s plasmid: Extract the plasmid from the CRISPRi-2s vector to obtain the CRISPRi-2s plasmid.
[0022] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, in step S2.2 or step S3.2, use T4 DNA ligase to ligate the vector with the gRNA sequence.
[0023] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, step S4 includes the following steps:
[0024] S4.1. Pick colonies from the basal strain and statically culture to obtain a bacterial solution;
[0025] S4.2. Suspend the bacterial solution in MgCl2 buffer to obtain a suspended bacterial solution;
[0026] S4.3. Add the CRISPR activation system, the CRISPR interference system, and an equal volume of glycerol solution to the suspended bacterial solution, then add an equal volume of glycerol solution, mix evenly to obtain a mixed solution;
[0027] S4.4. Electrotransform the mixed solution using an electroporator.
[0028] S4.5. Static culture the electrotransformed bacterial solution to obtain a new type of Schizosaccharomyces pombe strain transfected with CRISPRa-VP64 and CRISPRi-2s.
[0029] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, step S4 specifically includes the following steps:
[0030] S4.1. Pick colonies that have grown once from the initial Bifidobacterium longum subsp. infantis, transfer them to an LB medium, and statically culture until the bacterial solution is in the logarithmic growth phase to obtain a statically cultured bacterial solution.
[0031] S4.2. Filter the statically cultured bacterial solution, wash it twice with MgCl2 buffer, and then suspend the bacterial solution in MgCl2 buffer to obtain a suspended bacterial solution.
[0032] S4.3. Add the CRISPRa-VP64 plasmid and the CRISPRi-2s plasmid to the suspended bacterial solution respectively, then add an equal volume of glycerol solution, and mix evenly to obtain a mixed solution.
[0033] S4.4. Electrotransform the mixed solution using an electroporator.
[0034] S4.5. Transfer the electrotransformed bacterial solution to an LB medium for static culture to obtain a new type of Schizosaccharomyces pombe strain transfected with CRISPRa-VP64 and CRISPRi-2s.
[0035] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, in step S4.4, the electrotransformation parameters of the electroporator are: voltage is 1.8 kV, capacitance is 25 μF, resistance is 200 Ω, and time constant is 4.5 ms.
[0036] In the aforementioned method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, the Bifidobacterium longum includes the long subsp., infantis subsp., suis subsp., swine-derived subsp., or adolescentis subsp.
[0037] A Schizosaccharomyces pombe strain prepared by the above method.
[0038] The present invention also discloses the application of the above-mentioned Schizosaccharomyces pombe strain in the preparation of cosmetics.
[0039] Compared with the prior art, the beneficial effects of the present invention are:
[0040] The present invention designs the gRNA sequences of glutamate decarboxylase GAD and succinic semialdehyde dehydrogenase SSADH, constructs the CRISPR activation system of GAD and the CRISPR interference system of SSADH, and uses electroporation technology to introduce these systems into the basic strain Bifidobacterium longum, regulating the expression of glutamate decarboxylase (GAD) and succinic semialdehyde dehydrogenase (SSADH) in Bifidobacterium longum, to obtain a new strain of Bifidobacterium bifidum.
[0041] This strain can efficiently synthesize γ-aminobutyric acid (GABA), a butyric acid derivative that has a positive impact on human skin and intestinal health. This strain can grow rapidly, has good production stability and long-term storage capacity. As a new type of Bifidobacterium bifidum product, this strain can be applied to multiple fields such as cosmetics, beverages, foods, and healthcare products.
[0042] The present invention uses newly synthesized Bifidobacterium bifidum to ferment and produce GABA with glucose as the substrate, reducing the production cost, synergistically enhancing the biological activity. Nucleotides, polypeptides and GABA in its fermentation products jointly regulate the TRPV1 receptor, reducing the sensitivity of neuroinflammation, and the repair efficiency is increased by 30%. At the same time, SOD enzyme and GABA synergistically scavenge free radicals, and the lipid peroxidation inhibition rate reaches 72%. Moreover, the polysaccharide component of Bifidobacterium bifidum itself can self-assemble to form nano-microcapsules to encapsulate GABA, improving the transdermal absorption rate and extending the stability, and being compatible with anhydrous formulations. Both Bifidobacterium bifidum and GABA are endogenous substances, and the incidence of combined use is <0.3%. At the same time, short-chain fatty acids are used to optimize the skin flora and inhibit the quorum sensing of pathogenic bacteria, and the improvement effect on inflammatory skin diseases such as acne is prominent, with significant safety and microecological regulation advantages.
[0043] Moreover, the transformation method of the present invention by gene editing technology has the characteristics of stable process, simple operation, high efficiency, precision, and controllability, can save the preparation time, reduce the preparation cost, increase the yield, avoid the use of chemical synthesis, does not introduce foreign genes, is non-toxic to organisms, and does not affect cell growth. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 It is a growth curve graph of four different strains of Bifidobacterium bifidum in a fermenter.
[0045] Figure 2 It is a graph of the γ-aminobutyric acid (GABA) production of four different strains of Bifidobacterium bifidum in a fermenter for 24 hours. DETAILED DESCRIPTION OF THE INVENTION
[0046] The following further illustrates the present invention in conjunction with examples, but it is not used as a basis for limiting the present invention.
[0047] Example 1:
[0048] A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology, taking Bifidobacterium longum subsp. infantis as an example, includes the following steps:
[0049] S1. Select strains and target genes: Use Bifidobacterium longum subsp. infantis as the base strain.
[0050] Use the DNA gene sequences of the inherently existing glutamate decarboxylase (GAD) and succinic semialdehyde dehydrogenase (SSADH) in Bifidobacterium longum subsp. infantis as the target genes, and the target gene sequences are shown in the following table:
[0051] Table 1. Target gene sequences
[0052]
[0053] S2. Construct the CRISPR activation system for glutamate decarboxylase, specifically:
[0054] S2.1. Design the gRNA sequence for activating GAD: Select a group of gRNA sequences that match the PAM at the 3'-end NGG site of the target sequence in the GAD gene sequence from the GAD RNA sequence, which are:
[0055] 5'-CACCGGGAGGCGGAGGCGGAGCGTG-3' (upstream primer) and 5'-AAACCACGCTCCGCCTCCTCCGCCTCC-3' (downstream primer);
[0056] Synthesize and use PCR to amplify the above gRNA sequence to obtain the GAD-gRNA sequence.
[0057] S2.2. Clone the gRNA into the CRISPRa vector: Select pcDNA-dCas9-VP64 as the vector and linearize it, and use T4 DNA ligase to ligate the linearized pcDNA-dCas9-VP64 vector with the GAD-gRNA sequence to obtain the complete CRISPRa-VP64 vector with navigation function.
[0058] S2.3. Extract the CRISPRa-VP64 plasmid: Extract the plasmid from the CRISPRa-VP64 vector to obtain the CRISPRa-VP64 plasmid, which is the CRISPR activation system for GAD.
[0059] S3. Construct the CRISPR interference system for succinic semialdehyde dehydrogenase, specifically:
[0060] S3.1. Design gRNA sequences that inhibit SSADH: From the RNA sequence of SSADH, select a group of gRNA sequences that match the PAM located at the NGG site at the 3' end of the target sequence in the SSADH gene sequence, which are:
[0061] 5'-CACCGCAGACAGGAGCCAGCCGAGC-3' (upstream primer), 5'-AAACGCTCGGCTGGCTCCTGTCTGC-3' (downstream primer);
[0062] Synthesize and amplify the above gRNA sequences using PCR to obtain the SSADH-gRNA sequences;
[0063] S3.2. Clone gRNA into the CRISPRa vector: Select pCBH-CRISPRi-2s as the vector and linearize it. Use T4 DNA ligase to ligate the linearized pCBH-CRISPRi-2s vector with the SSADH-gRNA sequences to obtain the CRISPRi-2s vector.
[0064] S3.3. Extract the CRISPRi-2s plasmid: Extract the plasmid from the CRISPRi-2s vector to obtain the CRISPRi-2s plasmid, which is the CRISPR interference system for SSADH.
[0065] S4. Use the method of electroporation to introduce both the CRISPRa-VP64 plasmid and the CRISPRa-2s plasmid into Bifidobacterium longum subsp. infantis to obtain the new Saccharomyces boulardii strain BL-12.
[0066] Specifically:
[0067] S4.1. Pick colonies of the first growth from the initial Bifidobacterium longum subsp. infantis, transfer them to 10 ml of LB medium, and statically culture at 37°C until the bacterial solution is in the logarithmic growth phase (OD600 = 0.5 - 0.8) to obtain the statically cultured bacterial solution.
[0068] The formula of the LB medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1 liter of water. The sterilization method of the LB medium is: first adjust the pH value to 7.0 - 7.5, and then autoclave at 121°C for 15 - 20 minutes.
[0069] S4.2. Filter the statically cultured bacterial solution with sterile gauze, wash it twice with 0.1M MgCl2 buffer, and then suspend the bacterial solution in 0.1M MgCl2 buffer to obtain the suspended bacterial solution.
[0070] S4.3. Add 100 ng of CRISPRa-VP64 plasmid and 2 μg of CRISPRi-2s plasmid into the suspended bacterial solution respectively, then add an equal volume of 1 M glycerol solution, mix well to obtain a mixed solution.
[0071] S4.4. Transfer the mixed solution into an electroporator and perform electroporation according to the following parameters: voltage is 1.8 kV, capacitance is 25 μF, resistance is 200 Ω, and time constant is 4.5 ms.
[0072] S4.5. Transfer the electroporated bacterial solution into an LB medium at 37 °C and statically culture for 2 hours, then expand the culture at an appropriate dilution multiple to obtain a new type of Schizosaccharomyces pombe strain BL-12 transfected with CRISPRa-VP64 and CRISPRi-2s.
[0073] Comparative Example 1:
[0074] This comparative example is basically the same as the example, the difference is: the CRISPRa-VP64 plasmid was separately introduced into Bifidobacterium longum subsp. infantis to obtain a new type of Schizosaccharomyces pombe strain BL-64 transfected with CRISPRa-VP64.
[0075] Comparative Example 2:
[0076] This comparative example is basically the same as the example, the difference is: the CRISPRi-2s plasmid was separately introduced into Bifidobacterium longum subsp. infantis to obtain a new type of Schizosaccharomyces pombe strain BL-2s transfected with CRISPRi-2s.
[0077] Fermentation tank growth and production experiments of different Schizosaccharomyces pombe strains:
[0078] Prepare the medium: Use LB medium and prepare it according to the standard instructions, adjust the pH value to 6.2.
[0079] Strain pretreatment: Take four strains, namely the initial Bifidobacterium longum subsp. infantis strain (labeled as Wild), the BL-VP64 strain of Comparative Example 1, the BL-2s strain of Comparative Example 2, and BL-12 of Example 1, inoculate them into the LB medium, and let them stand overnight in a constant temperature incubator at 37 °C.
[0080] Prepare the fermentation tank: Sterilize the fermentation tank using an autoclave and fill 500 ml of LB medium into the fermentation tank.
[0081] Inoculate the strain: Transfer the strain into the fermentation tank using a sterile pipette, with an initial cell density of OD600 = 0.05.
[0082] Fermentation culture: Place the fermenter in a 37°C constant temperature incubator. At the same time, control the rotation speed in the fermenter at 100 rpm. Control the pH value in the fermenter at around 6.2 by adding hydrochloric acid and sodium hydroxide. Feed glucose at a rate of 10 g / L-hr and monitor the growth of the bacterial population during the culture process.
[0083] Throughout the 24-hour culture time, measure the absorbance at a wavelength of 600 nm, that is, the OD value, at regular intervals. The OD value is used to evaluate the measure of the growth of the bacterial population, and the OD value is proportional to the cell concentration. The specific detection data is shown in Figure 1 as follows.
[0084] From Figure 1 it can be seen that under the condition of using the same culture medium and the same culture conditions, different strains have different growth concentrations. Among them, BL-12 in Example 1 has the highest strain concentration, and its highest OD is about 36, indicating that the growth rate of the bacterial population of BL-12 is the fastest.
[0085] Analysis of GABA content in the fermentation broth:
[0086] Sample preparation: Take 1 mL of the fermentation broth in the fermenter, centrifuge at 12000 rpm for 10 minutes, take the supernatant, filter the sample using a 0.22 μm filter to remove microorganisms and impurities, and store the filtered liquid sample in a 1.5 mL centrifuge tube for HPLC analysis.
[0087] Chromatographic conditions: Use reverse-phase high-performance liquid chromatography (RP-HPLC) for analysis. Use a C18 chromatographic column, the mobile phase is methanol / water (40 / 60, volume ratio), and the flow rate is 1 mL / min. The detection wavelength is 210 nm.
[0088] Sample injection: Use an automatic injector to inject 10 μL of the sample into the chromatographic column, and avoid air entering the injector to prevent foam formation.
[0089] Data analysis: Prepare a standard curve according to the standard product with a known concentration, analyze the sample with an unknown concentration using the same chromatographic conditions, and calculate the concentration of GABA through the standard curve. The experimental results of the concentration of GABA after 24-hour culture are shown in Figure 2 as follows. From Figure 2 it can be seen that under the condition of using the same culture medium, different strains produce different concentrations of GABA. Among them, the GABA content produced by the strain BL-12 is the highest, which is 11.2 g / L, that is, the strain BL-12 is the best Saccharomyces boulardii strain for fermentative production of GABA.
[0090] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Those skilled in the art can modify the technical solutions recorded in the above embodiments or perform equivalent replacements for some of the technical features; and all such modifications and replacements should fall within the protection scope of the appended claims of the present invention.
Claims
1. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technique, characterized in that: It includes the following steps: S1. Select strains and target genes: Use Bifidobacterium longum of Schizosaccharomyces pombe as the basic strain, and use the DNA gene sequences of glutamate decarboxylase and succinic semialdehyde dehydrogenase in the basic strain as the target genes; S2. Construct the CRISPR activation system for glutamate decarboxylase to obtain the CRISPRa-VP64 plasmid; S3. Construct the CRISPR interference system for succinic semialdehyde dehydrogenase to obtain the CRISPRi-2s plasmid; S4. Use the method of electroporation to introduce both the CRISPRa-VP64 plasmid and the CRISPRi-2s plasmid into the basic strain to obtain a new Schizosaccharomyces pombe strain.
2. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology according to claim 1, characterized in that: Step S2 specifically includes the following steps: S2.1 Design the gRNA sequence for activating glutamate decarboxylase: From the RNA sequence of glutamate decarboxylase, select a group of gRNA sequences that match the PAM at the NGG site at the 3'-end of the target sequence in the glutamate decarboxylase gene sequence, then synthesize and amplify using PCR to obtain the GAD-gRNA sequence; S2.2 Clone the gRNA into the CRISPRa vector: Select pcDNA-dCas9-VP64 as the vector, connect the pcDNA-dCas9-VP64 vector with the GAD-gRNA sequence to obtain the CRISPRa-VP64 vector; S2.3 Extract the CRISPRa-VP64 plasmid: Extract the plasmid from the CRISPRa-VP64 vector to obtain the CRISPRa-VP64 plasmid.
3. A method for preparing Saccharomyces boulardii using the Crispr-cas gene editing technology according to claim 1, characterized in that: Step S3 specifically includes the following steps: S3.1 Design the gRNA sequence for inhibiting succinic semialdehyde dehydrogenase: From the RNA sequence of succinic semialdehyde dehydrogenase, select a group of gRNA sequences that match the PAM at the NGG site at the 3'-end of the target sequence in the succinic semialdehyde dehydrogenase gene sequence, then synthesize and amplify using PCR to obtain the SSADH-gRNA sequence; S3.2 Clone the gRNA into the CRISPRa vector: Select pCBH-CRISPRi-2s as the vector, connect the pCBH-CRISPRi-2s vector with the SSADH-gRNA sequence to obtain the CRISPRi-2s vector; S3.3 Extract the CRISPRi-2s plasmid: Extract the plasmid from the CRISPRi-2s vector to obtain the CRISPRi-2s plasmid.
4. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology according to claim 2 or 3, characterized in that: In step S2.2 or step S3.2, use T4 DNA ligase to connect the vector with the gRNA sequence.
5. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology according to claim 1, characterized in that: Step S4 includes the following steps: S4.1 Pick colonies from the basic strain and perform static culture to obtain a bacterial solution; S4.2 Suspend the bacterial solution in MgCl2 buffer to obtain a suspended bacterial solution; S4.3 Add the CRISPR activation system and the CRISPR interference system and an equal volume of glycerol solution to the suspended bacterial solution, then add an equal volume of glycerol solution, mix evenly to obtain a mixed solution; S4.4 Perform electroporation on the mixed solution using an electroporator; S4.
5. Static culture the electrotransformed bacterial solution to obtain a new type of Schizosaccharomyces pombe strain transfected with CRISPRa-VP64 and CRISPRi-2s.
6. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technology according to claim 5, characterized in that: Step S4 specifically includes the following steps: S4.
1. Pick colonies that have grown once from the initial Bifidobacterium longum subsp. infantis, transfer them to an LB medium, and statically culture until the bacterial solution is in the logarithmic growth phase to obtain a statically cultured bacterial solution. S4.
2. Filter the statically cultured bacterial solution, wash it twice with a MgCl2 buffer solution, and then suspend the bacterial solution in the MgCl2 buffer solution to obtain a suspended bacterial solution. S4.
3. Add the CRISPRa-VP64 plasmid and the CRISPRi-2s plasmid to the suspended bacterial solution respectively, and then add an equal volume of glycerol solution, mix evenly to obtain a mixture. S4.
4. Use an electroporator to electrotransform the mixture. S4.
5. Transfer the electrotransformed bacterial solution to an LB medium for static culture to obtain a new type of Schizosaccharomyces pombe strain transfected with CRISPRa-VP64 and CRISPRi-2s.
7. A method for preparing Schizosaccharomyces pombe using the Crispr-cas gene editing technique according to claim 5, characterized in that: In step S4.4, the electrotransformation parameters of the electroporator are: voltage is 1.8 kV, capacitance is 25 μF, resistance is 200 Ω, and time constant is 4.5 ms.
8. A method for preparing Saccharomyces boulardii using the Crispr-cas gene editing technique according to claim 1, characterized in that: The Bifidobacterium longum includes the subspecies longum, infantis, suis, suis-derived, or adolescentis.
9. A Saccharomyces boulardii strain, characterized in that: Prepared by the method according to any one of claims 1-7.
10. Use of the Schizosaccharomyces pombe strain according to claim 8 in the preparation of cosmetics.