Saccharomyces boulardii mevalonic acid high-efficiency production strain constructed based on genetic engineering optimization strategy and application thereof
Through genetic engineering optimization strategies, a yeast yeast strain that efficiently produces mevalonate was constructed, which solved the problem of low expression efficiency of existing strains, achieved the efficient production of mevalonate and improved ovarian function, and provided a new way for drug development.
Patent Information
- Application Number
- CN202510183186.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-16
AI Technical Summary
The existing yeast Brahmadria strains have low expression efficiency on the mevalonate synthesis pathway and cannot meet the growing market demand and efficient production requirements in specific application scenarios.
Through genetic engineering optimization strategies, a highly efficient production strain of yeast Brahmab was constructed. The specific methods include the use of seamless cloning technology to construct plasmids that overexpress tHMG1, knock out ROX1 and inhibit ERG9, and transform these plasmids into Brahmab through chemical transformation.
The efficient production of mevalonate was achieved. The mevalonate yield of the bacterial strain was significantly increased under the conditions of shake flask fermentation, reaching 2.3g/L. In animal experiments, the oocyte quality of aging ovaries was significantly improved, providing new ideas for the development of drugs to improve the reproductive health of elderly women.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biosynthesis application and discloses a Saccharomyces boulardii mevalonate efficient production strain constructed based on a genetic engineering optimization strategy and an application thereof. Background Art
[0002] The ovaries play an extremely important role in women's life. They are not only responsible for producing eggs, providing the possibility of nurturing new life, but also secreting a variety of hormones to maintain women's physiological characteristics and the stability of the body's internal environment. With age, the number of primordial follicles in the ovaries gradually decreases, and the quality of the eggs also declines. This is a natural physiological process that leads to a decline in ovarian function. Generally speaking, women's ovarian function begins to show obvious signs of decline after the age of 35.
[0003] Mevalonic acid, also known as mevalonic acid or mevalonic acid, is an organic compound that is of great significance in biochemistry and metabolic engineering. Mevalonic acid is a key metabolic intermediate in the mevalonate pathway, and the mevalonate metabolic pathway is widely present in higher eukaryotes and some microorganisms. At the same time, mevalonic acid is also an important precursor for the synthesis of cholesterol, terpenoids and other prenylated organisms. Studies have shown that the MVA pathway in granulosa cells is a key regulator of oocyte meiosis and euploidy, while aging-related abnormalities in the MVA pathway can lead to oocyte meiotic defects and aneuploidy. In vitro supplementation of MVA and its derivative isoprene compound geranylgeraniol can improve meiotic defects and aneuploidy in oocytes of aged mice.
[0004] At present, the production methods of mevalonic acid are mainly divided into two categories: chemical synthesis and biosynthesis. The chemical synthesis method faces the problems of scarce raw materials, high structural complexity and high cost, and the products are mostly racemic mixtures, which limits its applicability for large-scale production. In contrast, the biosynthesis method uses microorganisms such as Escherichia coli as a production platform, integrates the key genes of the mevalonic acid synthesis pathway into microorganisms through genetic engineering technology, and realizes large-scale production with the help of its efficient genetic operating system. This method is not only suitable for large-scale manufacturing, but also has significant advantages in product quality control. Typical industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae and Pseudomonas, have been used in the production of MVA.
[0005] Saccharomyces cerevisiae is widely used to produce a variety of terpenoids, including mevalonate, due to its clear genetic background, easy operation, and robust metabolism. Figure 1The diagram shows the metabolic pathway of mevalonic acid fermented by yeast in nature. Mevalonic acid is expensive and needs to be stored in a low temperature, dark and dry environment. It is easily degraded or inactivated by factors such as light, heat and humidity. Saccharomyces boulardii is a subspecies of Saccharomyces cerevisiae. Compared with Saccharomyces cerevisiae, Saccharomyces boulardii, as a probiotic, has a high degree of safety and tolerance in the human body and can reduce side effects during drug delivery. Secondly, the intestinal colonization ability of Saccharomyces boulardii contributes to the stable release and absorption of drug molecules in the intestine, thereby improving the bioavailability and efficacy of drugs. Using Saccharomyces boulardii as a chassis organism, high expression of mevalonic acid is achieved through genetic engineering technology. This innovative strategy provides a simple, convenient and efficient new way for probiotic drug delivery. As a widely recognized probiotic, Saccharomyces boulardii has excellent intestinal colonization ability and stability, which makes it an ideal carrier for drug delivery.
[0006] However, although Saccharomyces boulardii has shown significant advantages in drug delivery and probiotic applications, its potential in the efficient production of mevalonic acid has not been fully explored. Existing Saccharomyces boulardii strains have low expression efficiency in the mevalonic acid synthesis pathway and cannot meet the growing market demand and the requirements for efficient production in specific application scenarios. Summary of the invention
[0007] To solve the above problems, the present invention discloses an efficient mevalonate production strain of Saccharomyces boulardii constructed based on a genetic engineering optimization strategy and its application. The efficient mevalonate production strain of Saccharomyces boulardii constructed by the present invention not only provides a new solution for the large-scale production of mevalonate, but also provides a basis for the development of new drugs based on mevalonate to improve ovarian function.
[0008] The present invention includes the following technical solutions:
[0009] A Saccharomyces boulardii mevalonate efficient production strain constructed based on a genetic engineering optimization strategy, wherein the Saccharomyces boulardii mevalonate efficient production strain has a chromosome-integrated expression-enhanced HMG-CoA reductase encoding gene tHMG1, and simultaneously lacks a transcriptional repressor encoding gene ROX1 of a mevalonate pathway and a sterol biosynthesis pathway, and an endogenous sterol biosynthesis gene ERG9;
[0010] The nucleotide sequence of tHMG1 is shown in SEQ ID No. 1;
[0011] The nucleotide sequence of ROX1 is shown in SEQ ID No. 2;
[0012] The nucleotide sequence of ERG9 is shown in SEQ ID No.3.
[0013] The present invention also discloses a method for constructing the above-mentioned Saccharomyces boulardii mevalonate efficient production strain constructed based on genetic engineering optimization strategy, comprising the following steps:
[0014] S1 used seamless cloning technology to construct plasmids for overexpressing tHMG1, knocking out ROX1, and inhibiting ERG9;
[0015] S2 sequentially transforms the three plasmids in step S1 into Saccharomyces boulardii carrying the Cas9 plasmid by chemical transformation;
[0016] S3 identifies and selects positive transformants on the screening plate;
[0017] S4 was verified by colony PCR and sequencing, and an efficient mevalonate-producing strain of Saccharomyces boulardii was obtained.
[0018] Furthermore, in the above construction method, the plasmid for overexpressing tHMG1, the plasmid for knocking out ROX1, and the plasmid for inhibiting ERG9 in step S1 are all constructed based on the pYES2 plasmid.
[0019] The invention also discloses application of the Saccharomyces boulardii mevalonate efficient production strain in producing mevalonate.
[0020] Furthermore, in the above applications, the applications include:
[0021] 1) activating a Saccharomyces boulardii mevalonate efficient producing strain;
[0022] 2) The single colony obtained after inoculation and activation is placed in YPD medium for seed culture;
[0023] 3) The obtained seed culture solution is inoculated into YPD medium for fermentation to produce mevalonic acid.
[0024] The present invention also discloses the use of the Saccharomyces boulardii mevalonate efficient producing strain in the present invention in preparing a medicine for improving the quality of aged ovarian oocytes.
[0025] The present invention also discloses a medicine capable of improving the quality of aged ovarian oocytes, wherein the medicine comprises the boulardii mevalonate efficient producing strain of Saccharomyces boulardii of the present invention.
[0026] Furthermore, the above-mentioned medicine is an oral preparation of physiological saline dissolved with a high-efficiency mevalonate-producing strain of Saccharomyces boulardii.
[0027] Furthermore, in the above-mentioned medicine, the cell concentration of the Saccharomyces boulardii mevalonate efficient producing strain in the said physiological saline oral preparation is OD 600 =100.
[0028] The present invention has the following beneficial effects:
[0029] The present invention discloses a Saccharomyces boulardii mevalonate efficient production strain constructed based on a genetic engineering optimization strategy and its application, which has the following advantages:
[0030] 1. Efficient production of mevalonic acid: Through genetic engineering optimization strategies, a Saccharomyces boulardii strain capable of efficiently producing mevalonic acid was successfully constructed. Under shake flask fermentation conditions, the strain significantly increased the mevalonic acid production to 2.3 g / L, and the content in the bacteria was also significantly increased, providing an efficient microbial platform for large-scale production of mevalonic acid.
[0031] 2. Improve ovarian function: The mevalonic acid product of the strain of the present invention showed significant physiological activity in animal experiments. Oral administration to elderly female mice can significantly improve the quality of oocytes in aging ovaries, increase ovarian weight, ovarian index and the number of follicles at all levels, providing a new idea for the development of drugs to improve the reproductive health of elderly women.
[0032] 3. Enhanced drug delivery effect: As a probiotic, Saccharomyces boulardii has a high degree of safety and tolerance in the human body. The mevalonate-producing strain constructed with this as the base organism can not only improve the bioavailability and efficacy of the drug, but also reduce the side effects during drug delivery, providing a new way for probiotic drug delivery.
[0033] 4. Expanding the scope of application: The Saccharomyces boulardii mevalonate efficient production strain of the present invention can not only be used for the production of mevalonate, but also can be used as a platform strain to produce other physiologically active terpenoid compounds through further genetic engineering modification, and has broad application prospects.
[0034] 5. Cost reduction and environmental protection: Compared with chemical synthesis, biosynthesis uses microbial fermentation to produce mevalonate, which has the advantages of a wide source of raw materials, low production costs, and environmental friendliness, and is in line with the concept of sustainable development.
[0035] In summary, the Saccharomyces boulardii mevalonate efficient production strain constructed by genetic engineering in the present invention shows significant beneficial effects in mevalonate production, drug development and probiotic application, and has important scientific value and socio-economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is a diagram of the metabolic pathway of mevalonic acid fermentation by yeast in nature;
[0037] Figure 2 It is a schematic diagram of the fermentation yield of mevalonic acid of the recombinant Saccharomyces boulardii strain with high mevalonic acid production of the present invention and the control strain;
[0038] Figure 3 It is the structure map of pYES2-hyg and pHCas9 plasmid;
[0039] Figure 4 The in vivo administration significantly improved the ovarian function of aged female mice;
[0040] Figure 5 The figure shows that in vivo administration significantly improves the number of follicles in aged female mice. DETAILED DESCRIPTION
[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0042] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.
[0043] Materials and Methods
[0044] LB medium (for DH5α growth): 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, sterilized at 121°C for 20 min; if prepared as solid culture medium, add 15-20 g / L agar powder.
[0045] YPD medium (for the growth of Saccharomyces boulardii): 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, sterilized at 115°C for 20 min; if prepared as a solid culture medium, add 15-20 g / L agar powder.
[0046] Table 1 PCR amplification system
[0047]
[0048] Table 2 PCR amplification conditions
[0049]
[0050] The sequences involved in the present invention are as follows:
[0051] SEQ ID No.1
[0052]
[0053]
[0054] SEQ ID No.2
[0055]
[0056]
[0057] SEQ ID No.3
[0058]
[0059]
[0060] SEQ ID No.4
[0061]
[0062] SEQ ID No.5
[0063]
[0064]
[0065] SEQ ID No.6
[0066]
[0067] SEQ ID No.7
[0068]
[0069]
[0070] SEQ ID No.8
[0071]
[0072] SEQ ID No.9
[0073]
[0074] SEQ ID No.10
[0075]
[0076]
[0077] SEQ ID No.11
[0078]
[0079] SEQ ID No.12
[0080]
[0081]
[0082] SEQ ID No.13
[0083]
[0084] The remaining primer sequences are shown in Table 3, and are numbered as SEQ ID NO. 14-37 from top to bottom.
[0085] Table 3 Primer table
[0086]
[0087]
[0088]
[0089] In the following examples, the competent transformation, culture and storage methods of DH5α are as follows:
[0090] 1. Transformation: Add the transformation system to the DH5α competent medium melted on ice, ice bath for 30 minutes, heat shock at 42℃ for 60 seconds, quickly cool on ice for 3-5 minutes, add 1ml LB liquid culture medium to the tube, mix well and culture at 37℃ for 1 hour, mix the above bacterial solution and spread 100μl on the screening plate containing Amp, and culture at 37℃ for 12-16h.
[0091] 2. Cultivation: Pick the strain and streak or spread it on the solid LB plate with the corresponding resistance, then invert and culture it in a constant temperature incubator at 37°C for 12-16 hours. Pick the freshly recovered E. coli monoclone from the LB solid culture medium plate, inoculate it into a certain volume of LB liquid culture medium, and add the corresponding resistance as needed, and culture it at 37°C and 220rpm for 12-16 hours;
[0092] 3. Storage: Take 500 μL of fresh overnight culture seed solution, add it to a cryovial containing 500 μL of 50% sterilized glycerol, and store it in a -80℃ ultra-low temperature refrigerator;
[0093] The pHCas9 plasmid (pHcas9-nours) of the present invention is shown in Figure 3 As shown in a in the figure, the map of the pYES2 plasmid (pYES2-gRNA-hyg) in the present invention is shown in Figure 3 As shown in b.
[0094] Example 1
[0095] The method for constructing a recombinant Saccharomyces boulardii ESB001 that highly expresses mevalonic acid specifically comprises the following steps:
[0096] 1.1 Using pYES2 plasmid (empty vector) as template, perform inverse PCR amplification with primers pYES2-F and pYES2-R and gel recovery to obtain linearized pYES2 fragment.
[0097] 1.2 Using pYES2 plasmid (empty vector) as a template, primers gRNA1-F and gs-R were used for amplification and gel recovery to obtain fragment 1 (SEQ ID No. 4) containing gRNA.
[0098] 1.3 Using the genome of Saccharomyces boulardii as a template, primers PT1-F, PT1-R, HP-F, HP-R and HA-F, HA-R were used to amplify and gel-recovery to obtain tHMG1, promoter PGK1 and terminator ADH1, and primers HP-F and HA-R were used for fusion PCR and gel-recovery to obtain PGK1-tHMG1-ADH1 fragment 2 (SEQ ID No. 5).
[0099] 1.4 Using the genome of Saccharomyces boulardii as a template, primers HO-UP-F, HO-UP-R and primers HO-DOWN-F, HO-DOWN-R were used to amplify the 1000 bp region before and after the HO gene coding region and recovered from the gel to obtain fragments 3 (SEQ ID No. 6) and 4 (SEQ ID No. 7).
[0100] 1.5 Fragment 1, fragment 2, fragment 3, fragment 4 and linearized pYES2 fragment were connected using ClonExpress UltraOne Step Cloning Kit to obtain recombinant plasmid pYES2-tHMG1.
[0101] 1.6 First, the pHCas9 plasmid (Nanjing GenScript Company) was transformed into Saccharomyces boulardii (CNCM1-745, Biokeda Pharmaceuticals, France), and then the recombinant plasmid pYES2-tHMG1 in step 1.5 was transferred into the Saccharomyces boulardii containing the pHCas9 plasmid. According to the instructions of the yeast transformation kit, the yeast cells were inoculated into 3 mL YPD liquid medium for activation, and then transferred to new YPD medium and cultured until OD 600=0.6, centrifuge and discard the supernatant, add sterile deionized water to wash, add 1× lithium acetate solution and centrifuge and discard the supernatant, add competent freezing solution to resuspend, and the yeast competent cells are ready. 310μL premix is composed of 240μL PEG Solution, 36μL 10×LiAc Solution, 10μL Carrier DNA, 5μL plasmid and 19μL sterile deionized water. The premix is added to 50μL competent cells and mixed, incubated in a 30℃ water bath for 30min, mixed every 10min, centrifuged and discarded the supernatant, resuspended with 1mL YPD, cultured on a shaker at 30℃ for 30-60min, centrifuged and discarded the supernatant, added 200μL sterile deionized water to resuspend, and 100μL is coated on a YPD plate containing hygromycin and nouril mycelial resistance. A single colony was picked on the resistance plate, and the positive transformant was identified by colony PCR verification and sequencing. The identified positive transformant was the recombinant Saccharomyces boulardii ESB001.
[0102] The method for constructing a recombinant Saccharomyces boulardii ESB002 that highly expresses mevalonic acid specifically comprises the following steps:
[0103] 2.1 Using pYES2 plasmid (empty vector) as template, reverse PCR amplification was performed with primers pYES2-F and pYES2-R and gel recovery was performed to obtain the linearized pYES2 fragment.
[0104] 2.2 Using pYES2 plasmid (empty vector) as a template, primers gRNA2-F and gs-R were used for amplification and gel recovery to obtain fragment 5 (SEQ ID No. 8) containing gRNA.
[0105] 2.3 Using the genome of Saccharomyces boulardii as a template, primers ROX1-UP-F, ROX1-UP-R and primers ROX1-DOWN-F, ROX1-DOWN-R were used to amplify the 900 bp region before and after the ROX1 gene coding region and gel-recovered to obtain fragments 6 (SEQ ID No. 9) and 7 (SEQ ID No. 10).
[0106] 2.4 Fragment 5, fragment 6, fragment 7 and linearized pYES2 fragment were connected using ClonExpress Ultra One Step Cloning Kit to obtain recombinant plasmid pYES2-ROX1.
[0107] 2.5 First, the pHCas9 plasmid was transformed into the recombinant Saccharomyces boulardii ESB001, and then the recombinant plasmid pYES2-ROX1 in step 2.4 was transferred into the recombinant Saccharomyces boulardii ESB001 containing the pHCas9 plasmid. Single colonies were selected on YPD plates containing hygromycin and nouril resistance, and positive transformants were identified by colony PCR verification and sequencing. The identified positive transformants were recombinant Saccharomyces boulardii ESB002.
[0108] The method for constructing a recombinant Saccharomyces boulardii ESB003 that highly expresses mevalonic acid specifically comprises the following steps:
[0109] 3.1 Using pYES2 plasmid (empty vector) as template, reverse PCR amplification was performed with primers pYES2-F and pYES2-R and gel recovery was performed to obtain the linearized pYES2 fragment.
[0110] 3.2 Using pYES2 plasmid (empty vector) as a template, primers gRNA3-F and gs-R were used for amplification and gel recovery to obtain fragment 8 (SEQ ID No. 11) containing gRNA.
[0111] 3.3 Using the genome of Saccharomyces boulardii as a template, the promoter of the ERG9 gene was amplified with primers ERG9p-UP-F, ERG9p-UP-R and primers ERG9p-DOWN-F, ERG9p-DOWN-R to truncate the 1000 bp region before and after the coding region, and fragments 9 (SEQ ID No. 12) and 10 (SEQ ID No. 13) were obtained by gel recovery.
[0112] 3.4 Fragment 8, fragment 9, fragment 10 and linearized pYES2 fragment were connected using ClonExpress Ultra OneStep Cloning Kit to obtain recombinant plasmid pYES2-ERG9p.
[0113] 3.5 The pHCas9 plasmid was first transformed into the recombinant Saccharomyces boulardii ESB002, and then the recombinant plasmid pYES2-ERG9p in step 3.4 was transformed into the recombinant Saccharomyces boulardii ESB002 containing the pHCas9 plasmid, and the steps were the same as 1.6 in Example 1. Single colonies were selected on a YPD plate containing hygromycin and nouril filament resistance, and positive transformants were identified by colony PCR and sequencing. The identified positive transformants were recombinant Saccharomyces boulardii ESB003.
[0114] Example 2
[0115] The use of engineered Saccharomyces boulardii (recombinant Saccharomyces boulardii ESB003 in Example 1) to ferment and produce mevalonic acid comprises the following steps:
[0116] A single colony of the mevalonate-producing strain of Saccharomyces boulardii activated on the YPD plate was inoculated into 50 ml YPD liquid medium and cultured at 37°C and 220 rpm for 14 h. The colony was transferred to 50 ml YPD liquid medium at a 2% inoculum and cultured at 37°C and 220 rpm until OD 600 =0.6-0.8, obtain seed solution;
[0117] The obtained seed liquid was transferred to a 250 mL conical flask containing 50 mL of fermentation medium at a 5% inoculum amount, and cultured at 37°C and 220 rpm for 48 h to obtain a fermentation liquid containing mevalonic acid. The content of mevalonic acid was determined by LC-MS, as shown in FIG. Figure 2 As shown, the content of mevalonic acid in the original strain was 58.67 ng / OD, and the mevalonic acid production in the culture medium was 31.63 mg / L. The content of mevalonic acid in the engineered Saccharomyces boulardii strain was 200.51 ng / OD, and the mevalonic acid production in the culture medium reached 2.32 g / L, with a significant increase in yield.
[0118] Example 3
[0119] The present embodiment provides a drug that can improve the quality of aged ovarian oocytes. The drug is a normal saline oral preparation containing engineered Saccharomyces boulardii (the recombinant Saccharomyces boulardii ESB003 in Example 1). The preparation method is to dissolve the engineered Saccharomyces boulardii in normal saline to prepare a Saccharomyces boulardii oral solution with a concentration of 100OD, that is, a normal saline solution oral preparation of Saccharomyces boulardii engineered bacteria.
[0120] The oral preparation of physiological saline solution of the engineered bacteria Saccharomyces boulardii is used to improve the quality of aged ovarian oocytes.
[0121] (1) 9.5-month-old C57BL6 mice were divided into three groups: a control group (normal saline solution), a wild-type Saccharomyces boulardii administration group, and a Saccharomyces boulardii engineering bacteria administration group (oral preparation of normal saline solution of Saccharomyces boulardii engineering bacteria). The mice were given drugs at 16:00 every day. The control group, the wild-type Saccharomyces boulardii administration group, and the Saccharomyces boulardii engineering bacteria administration group were gavaged with 100 μL of OD 600 =100, administered continuously for 14 days.
[0122] (2) Weigh the mice in each group, kill them by dislocation, make a U-shaped incision in the lower abdomen, expose the gross visceral structure of the abdominal cavity, look for the double uterine body, and extend upward to look for the ovaries connected to the fallopian tubes. Remove the fat tissue around the ovaries under a stereoscope and take pictures. Place the photographed ovarian tissue on dry sterile gauze, and weigh it using an electronic analytical balance after no liquid remains. Calculate the ovarian index according to the following formula: Ovarian index (ovarian index) = ovarian weight (mg) / body weight (g) × 100%. After fixation and embedding, the ovarian tissue was serially sectioned and the number of follicles at each level was calculated after HE staining. The results are as follows. Figure 4 and Figure 5 As shown: Figure 4 a, b, and c are white light images of the ovaries of the control group, the wild-type Saccharomyces boulardii administration group, and the Saccharomyces boulardii engineered bacteria administration group, respectively. Figure 4 d are the ovarian index statistics of the control group, the wild-type Saccharomyces boulardii administration group and the Saccharomyces boulardii engineering bacteria administration group, Figure 5 a, b, and c are the HE staining results of ovarian sections of the control group, the wild-type Saccharomyces boulardii administration group, and the Saccharomyces boulardii engineering bacteria administration group, respectively. Figure 5 d are statistical graphs of the number of follicles in the control group, the wild-type Saccharomyces boulardii administration group, and the Saccharomyces boulardii engineered bacteria administration group.
[0123] As can be seen from the figure, administration of engineered Saccharomyces boulardii to aged mice for 14 days significantly increased the ovarian weight, ovarian index and the number of follicles at all levels.
[0124] Therefore, in vivo supplementation of the engineered Saccharomyces boulardii (the recombinant Saccharomyces boulardii ESB003 in Example 1) disclosed in the present invention can improve the ovarian reserve of aged mice, and further, the engineered Saccharomyces boulardii can be used to prepare drugs for improving the quality of aged ovarian oocytes.
[0125] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.
Claims
1. A highly efficient mevalonate producing strain of Saccharomyces boulardii constructed based on genetic engineering optimization strategy, characterized in that: The chromosome of the Saccharomyces boulardii mevalonate efficient production strain is integrated with the expression-enhanced HMG-CoA reductase encoding gene tHMG1, and the transcriptional repressor encoding gene ROX1 of the mevalonate pathway and sterol synthesis pathway and the endogenous sterol biosynthesis gene ERG9 are simultaneously lacking; The nucleotide sequence of tHMG1 is shown in SEQ ID No. 1; The nucleotide sequence of ROX1 is shown in SEQ ID No. 2; The nucleotide sequence of ERG9 is shown in SEQ ID No.
3.
2. The method for constructing a highly efficient mevalonate producing strain of Saccharomyces boulardii according to claim 1, characterized in that: The following steps are involved: S1 used seamless cloning technology to construct plasmids for overexpressing tHMG1, knocking out ROX1, and inhibiting ERG9; S2 sequentially transforms the three plasmids in step S1 into Saccharomyces boulardii carrying the Cas9 plasmid by chemical transformation; S3 identifies and selects positive transformants on the screening plate; S4 was verified by colony PCR and sequencing, and an efficient mevalonate-producing strain of Saccharomyces boulardii was obtained.
3. The construction method according to claim 2, characterized in that: The plasmid for overexpressing tHMG1, the plasmid for knocking out ROX1, and the plasmid for inhibiting ERG9 in step S1 are all constructed based on the pYES2 plasmid.
4. Use of the Saccharomyces boulardii mevalonate efficient producing strain according to claim 1 in producing mevalonate.
5. The use according to claim 4, characterized in that: The applications include: 1) activating a Saccharomyces boulardii mevalonate efficient producing strain; 2) The single colony obtained after inoculation and activation is placed in YPD medium for seed culture; 3) The obtained seed culture solution is inoculated into YPD medium for fermentation to produce mevalonic acid.
6. Use of the Saccharomyces boulardii mevalonate efficient producing strain according to claim 1 in the preparation of a drug for improving the quality of aged ovarian oocytes.
7. A drug for improving the quality of aged ovarian oocytes, characterized in that: The medicine comprises the Saccharomyces boulardii mevalonate efficient producing strain according to claim 1.
8. The drug according to claim 7, characterized in that The medicine is an oral preparation of physiological saline in which a high-efficiency mevalonate-producing strain of Saccharomyces boulardii is dissolved.
9. The drug according to claim 8, characterized in that The cell concentration of the Saccharomyces boulardii mevalonate efficient producing strain in the physiological saline oral preparation is OD 600 =100.