A method for enhancing the freezing tolerance of Saccharomyces cerevisiae

Abstract

The present invention provides a method for enhancing the freezing tolerance of Saccharomyces cerevisiae. By using a specific biological material to construct recombinant Saccharomyces cerevisiae and then inducing the expression of the recombinant Saccharomyces cerevisiae, the freezing tolerance of Saccharomyces cerevisiae can be effectively enhanced. The present invention discovers for the first time that by transferring a recombinant plasmid containing the MET17 gene into Saccharomyces cerevisiae, the resulting recombinant Saccharomyces cerevisiae has a significantly improved freezing tolerance compared to Saccharomyces cerevisiae, ensuring its normal fermentation growth in a frozen environment and significantly improving the quality of frozen products (such as foods, cosmetics, drugs, etc.).

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. More specifically, it relates to a method for enhancing the freezing tolerance of Saccharomyces cerevisiae. Background Art

[0002] Saccharomyces cerevisiae has poor freezing tolerance, mainly due to: (1) Ice crystal damage: During the freezing process, the water in the cells forms ice crystals, directly piercing the cell wall and cell membrane, resulting in the leakage of cell contents; (2) Stagnation of metabolic activities: Low temperature (<0 °C) will completely stop the metabolism of Saccharomyces cerevisiae, and long-term freezing leads to the loss of organelle functions, and it is difficult to restore normal fermentation ability even after thawing; (3) Osmotic pressure imbalance: Freezing will cause a drastic change in the distribution of water inside and outside the cells, resulting in osmotic pressure imbalance, and then causing cell dehydration or swelling and rupture.

[0003] Moreover, the low freezing tolerance of Saccharomyces cerevisiae will directly or indirectly affect its applications in food, cosmetics, and pharmaceuticals. For example: (1) When fermenting frozen dough, the activity of Saccharomyces cerevisiae will decrease, which may lead to the failure of dough proofing; (2) When brewing wine at low temperature, the metabolic rate of Saccharomyces cerevisiae will decrease, which may lead to an overly long fermentation cycle, low brewing efficiency, and at the same time increase the risk of contamination by miscellaneous bacteria, and the quality and stability of the finished wine will decline; (3) When preparing cosmetics and pharmaceuticals, freezing may damage the bioactive components in the extracts or fermentation products of Saccharomyces cerevisiae, resulting in a decline in product efficacy or stability.

[0004] Therefore, finding a method for enhancing the freezing tolerance of Saccharomyces cerevisiae is quite necessary for the fields of food, cosmetics, and pharmaceuticals. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention aims to provide a method for enhancing the freezing tolerance of Saccharomyces cerevisiae. By constructing recombinant Saccharomyces cerevisiae using specific biological materials and then inducing the expression of the recombinant Saccharomyces cerevisiae, the freezing tolerance of Saccharomyces cerevisiae can be effectively enhanced.

[0006] The first object of the present invention is to provide a method for enhancing the freezing tolerance of Saccharomyces cerevisiae.

[0007] The second object of the present invention is to provide a method for constructing recombinant Saccharomyces cerevisiae.

[0008] The third object of the present invention is to provide the recombinant Saccharomyces cerevisiae constructed by the above method.

[0009] The fourth object of the present invention is to provide the application of the above recombinant Saccharomyces cerevisiae in the preparation of products with high freezing tolerance.

[0010] The fifth object of the present invention is to provide the application of the MET17 gene or MET17 protein in enhancing the freezing tolerance of Saccharomyces cerevisiae.

[0011] The above object of the present invention is achieved by the following technical solutions:

[0012] The present invention provides a method for enhancing the freezing tolerance of Saccharomyces cerevisiae, that is: first construct recombinant Saccharomyces cerevisiae, and then induce the expression of the recombinant Saccharomyces cerevisiae;

[0013] Among them, the following one or several biological materials are used to construct recombinant Saccharomyces cerevisiae:

[0014] ①: The MET17 gene (the nucleotide sequence is as shown in SEQ ID NO: 15);

[0015] ②: The recombinant expression vector containing ①;

[0016] ③: The expression cassette containing ①;

[0017] ④: The recombinant expression vector containing ③.

[0018] Preferably, the recombinant expression vector is S.cry-EGFP-KanMX.

[0019] Preferably, the Saccharomyces cerevisiae is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-01 strain, deposited in the Guangdong Provincial Microbial Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0020] Preferably, the recombinant Saccharomyces cerevisiae is recombinant Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-03 strain, deposited in the Guangdong Provincial Microbial Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66060, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0021] Based on this, the present invention also provides a method for constructing recombinant Saccharomyces cerevisiae, that is: first construct a recombinant plasmid containing the MET17 gene, and then transfer it into Saccharomyces cerevisiae.

[0022] Preferably, the Saccharomyces cerevisiae is Saccharomyces cerevisiae LKF-01 strain, deposited in the Guangdong Provincial Microbial Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0023] The present invention for the first time discovers that transferring a recombinant plasmid containing the MET17 gene into Saccharomyces cerevisiae results in a significantly improved freezing tolerance of the obtained recombinant Saccharomyces cerevisiae compared to Saccharomyces cerevisiae, ensuring its normal fermentation growth in a frozen environment and significantly improving the quality of frozen products (such as foods, cosmetics, drugs, etc.). Therefore, the recombinant Saccharomyces cerevisiae constructed by the above method, the application of the above recombinant Saccharomyces cerevisiae in the preparation of products with high freezing tolerance, and the application of the MET17 gene or MET17 protein (amino acid sequence as shown in SEQ ID NO: 16) in enhancing the freezing tolerance of Saccharomyces cerevisiae should all be within the protection scope of the present invention.

[0024] Preferably, the recombinant Saccharomyces cerevisiae is the recombinant Saccharomyces cerevisiae strain LKF-03, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on March 26, 2025, with the deposit number GDMCC No: 66060, and the deposit address is on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0025] Preferably, the product is one or more of foods, cosmetics, and drugs.

[0026] Preferably, the Saccharomyces cerevisiae is the Saccharomyces cerevisiae strain LKF-01, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on March 26, 2025, with the deposit number GDMCC No: 66058, and the deposit address is on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0027] The present invention has the following beneficial effects:

[0028] The present invention for the first time discovers that transferring a recombinant plasmid containing the MET17 gene into Saccharomyces cerevisiae results in a significantly improved freezing tolerance of the obtained recombinant Saccharomyces cerevisiae compared to Saccharomyces cerevisiae, ensuring its normal fermentation growth in a frozen environment and significantly improving the quality of frozen products (such as foods, cosmetics, drugs, etc.). Description of the Drawings

[0029] Figure 1 It is the agarose gel electrophoresis pattern of plasmid S.cry-EGFP-KanMX in Example 1. Among them, "1" represents the PCR product band, and "M" represents the Marker band.

[0030] Figure 2 It is the agarose gel electrophoresis pattern of plasmid S.cry-TEF1-MET17-EGFP-KanMX in Example 1. Among them, "1" represents the PCR product band, and "M" represents the Marker band.

[0031] Figure 3Agarose electrophoresis diagram of Example 2. Among them, "1" represents the PCR product band, and "M" represents the Marker band.

[0032] Figure 4 Fluorescence microscope diagram of Example 2. Detailed implementation mode

[0033] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0035] Example 1 Construction of recombinant Saccharomyces cerevisiae

[0036] (1) Using plasmid pAUR123-EGFP as a template, the EGFP + terminator ADH1 fragment sequence was amplified with primers F1 / R1.

[0037] Among them, the primers are:

[0038] Primer F1 (SEQ ID NO:1): tctaacccgggtgatatcatggtgagcaagggcgagg;

[0039] Primer R1 (SEQ ID NO:2): ctgtcgattcgatactaacg.

[0040] The nucleotide sequence of the EGFP + terminator ADH1 fragment sequence is shown in SEQ ID NO:3:

[0041]

[0042] (2) Using the genomic DNA of Saccharomyces cerevisiae strain LKF-01 (deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on March 26, 2025, with the deposit number GDMCC No: 66058) as a template, the upstream rDNA homologous sequence was amplified with primers F2 / R2, the downstream rDNA homologous sequence was amplified with primers F3 / R3, the promoter TEF1 was amplified with primers F4 / R4, and the MET17 gene was amplified with primers F5 / R5.

[0043] Primer F2 (SEQ ID NO:4): ccggaacctctaatcattcgc;

[0044] Primer R2 (SEQ ID NO:5): gatatcacccgggttagagagctcgcaagtacggtcgttttaggttttacc;

[0045] Primer F3 (SEQ ID NO:6): actagtacgtctcgagattatacctcaagcacgcagagaa;

[0046] Primer R3 (SEQ ID NO:7): aacgaacgagaccttaacctactaaa;

[0047] Primer F4 (SEQ ID NO:8): aacgaccgtacttgcgagctcccacacaccatagcttcaaaatg;

[0048] Primer R4 (SEQ ID NO:9): ggcagaaccagcagtcatttagattagattgctatgctttctttctaa;

[0049] Primer F5 (SEQ ID NO:10): aaatggtactactatcggtggt;

[0050] Primer R5 (SEQ ID NO:11): gcccttgctcaccatgatatctggtttttggccagcgaaaac.

[0051] The nucleotide sequence of the upstream rDNA homologous sequence is shown in SEQ ID NO:12:

[0052]

[0053] The nucleotide sequence of the downstream rDNA homologous sequence is shown in SEQ ID NO: 13:

[0054]

[0055] The nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO:14:

[0056] ccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattccccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaatttttttttttgatttttttctctttcgatgacctcccattgatatttaagttattaaatggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa。

[0057] The nucleotide sequence of the MET17 gene is shown in SEQ ID NO:15, and the amino acid sequence encoded thereby is shown in SEQ ID NO:16.

[0058] SEQ ID NO:15:

[0059]

[0060] SEQ ID NO:16:

[0061] MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQLFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNIVSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNVPDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGIIVDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFASFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNGFGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQLNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP。

[0062] (3) Using plasmid pRCC-k as a template, the Amp+ori fragment sequence was amplified with primers F6 / R6, and the promoter TEF1+KanMX+terminator TEF1 fragment sequence was amplified with primers F7 / R7.

[0063] Primer F6 (SEQ ID NO:17): tttagtaggttaaggtctcgttcgttcacttttcggggaaatgtgc;

[0064] Primer R6 (SEQ ID NO:18): gcgaatgattagaggttccggtacctgctggcgtttttccatagg;

[0065] Primer F7 (SEQ ID NO:19): cgttagtatcgaatcgacagcagtatagcgaccagcattc;

[0066] Primer R7 (SEQ ID NO:20): ctgcgtgcttgaggtataatctcgagacgtactagttagggataacagggtaatcagcg.

[0067] The nucleotide sequence of the Amp+ori fragment is shown in SEQ ID NO:21:

[0068]

[0069] The nucleotide sequence of the promoter TEF1 + KanMX + terminator TEF1 fragment sequence is shown in SEQ ID NO: 22:

[0070]

[0071] (4) Recombine the upstream rDNA homologous sequence, the EGFP + terminator ADH1 fragment sequence, the promoter TEF1 + KanMX + terminator TEF1 fragment sequence, the downstream rDNA homologous sequence, and the Amp + ori fragment sequence to obtain the plasmid S.cry-EGFP-KanMX.

[0072] (5) Transfer the plasmid S.cry-EGFP-KanMX obtained in (4) into Escherichia coli DH5α by heat shock method, then coat it on an LB plate containing 100 μg / mL ampicillin (Amp), and culture it at 37 °C for 24 h. Then pick monoclonal bodies, use the monoclonal bodies as templates, use primer F2 (SEQ ID NO:4) and primer R3 (SEQ ID NO:7) as PCR verification primers for PCR verification, and then perform agarose gel electrophoresis on the PCR products. The results are as Figure 1 shown. It can be seen from the figure that the band of the PCR product is above 4000 bp, indicating that the plasmid S.cry-EGFP-KanMX is successfully constructed.

[0073] (6) After double digestion of the plasmid S.cry-EGFP-KanMX obtained in (4) with SmaI and EcoRV, recombine it with the promoter TEF1 and the MET17 gene to obtain the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX.

[0074] (7) Transfer the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX obtained in (6) into Escherichia coli DH5α by heat shock method, then coat it on an LB plate containing 100 μg / mL ampicillin (Amp), and culture it at 37 °C for 24 h. Then pick monoclonal bodies, use the monoclonal bodies as templates, use primer F4 (SEQ ID NO:8) and primer R5 (SEQ ID NO:11) as PCR verification primers for PCR verification, and then perform agarose gel electrophoresis on the PCR products. The results are as Figure 2 shown. It can be seen from the figure that the band of the PCR product is near 2000 bp, indicating that the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX is successfully constructed.

[0075] (8) Using the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX obtained in (6) as a template, and using primer F2 (SEQ IDNO:4) and primer R3 (SEQ ID NO:7) as PCR specific primers, amplify to obtain the rDNA up -TEF1-MET17-EGFP-KanMX-rDNA down sequence.

[0076] (9) The rDNA obtained in (8) up -TEF1-MET17-EGFP-KanMX-rDNA down was electrotransformed into the Saccharomyces cerevisiae strain LKF-01, and the recombinant Saccharomyces cerevisiae strain LKF-03 was constructed (deposited at the Guangdong Provincial Microbial Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66060).

[0077] Example 2 Screening of recombinant Saccharomyces cerevisiae

[0078] (1) The recombinant Saccharomyces cerevisiae strain LKF-03 was spread on a yeast extract peptone dextrose agar medium (YPD plate) containing 400 μg / mL geneticin (G418) and 1 mol / L sorbitol, and cultured at 30 °C for 72 h. Then, the transformants were picked and inoculated into 1 mL of yeast extract peptone dextrose medium (YPD liquid medium), and cultured with shaking at 30 °C and 180 rpm for 24 h to obtain a culture solution.

[0079] (2) The genomic DNA of the transformants in (1) was extracted, and using this genomic DNA as a template, primers F4 (SEQ ID NO:8) and primer R1 (SEQ ID NO:2) were used as PCR verification primers for PCR verification. Then, agarose gel electrophoresis was performed on the PCR products, and the results are as Figure 3 shown. As can be seen from the figure, the band of the PCR product is near 3000 bp, and sequencing confirmed that it contains the nucleotide sequence shown in SEQ ID NO:15, indicating that the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX has been successfully transferred into the Saccharomyces cerevisiae strain LKF-01, and the recombinant Saccharomyces cerevisiae strain LKF-03 has been successfully constructed.

[0080] (3) The culture solution obtained in (1) was observed with a fluorescence microscope (excitation wavelength of 465 - 495 nm, emission filter wavelength of 512 - 558 nm, using a 10× objective lens), and the fluorescence microscope image obtained is as Figure 4 shown. Since the recombinant plasmid S.cry-TEF1-MET17-EGFP-KanMX and the MET17 protein are fusion-expressed, and EGFP is a green fluorescent protein, the green fluorescence in the fluorescence microscope image can prove that the MET17 protein has been successfully expressed in the recombinant Saccharomyces cerevisiae strain LKF-03.

[0081] Example 3 Testing the freezing tolerance of recombinant Saccharomyces cerevisiae

[0082] I. Reagent preparation

[0083] Methylene blue staining solution: Mix 0.025 g of methylene blue, 0.048 g of calcium chloride hexahydrate, 1.0 g of glucose, 0.042 g of potassium chloride, and 0.02 g of sodium bicarbonate, and then make up the volume to 100 mL with sterile normal saline to obtain it.

[0084] II. Test method

[0085] (1) Inoculate the recombinant Saccharomyces cerevisiae LKF-03 strain and the Saccharomyces cerevisiae LKF-01 strain into yeast extract peptone dextrose medium (YPD liquid medium) containing 200 mg / mL of geneticin (G418) respectively, and shake culture at 30 °C and 180 rpm for 24 h. Then, aliquot 1 mL of the culture solution into each tube, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0086] (2) Place the precipitated cells obtained by centrifugation in a -20 °C refrigerator and store them for 1, 3, and 7 d respectively. Then take them out, thaw them at room temperature (25 °C), add 1 mL of PBS to resuspend the cells, and let them stand for 30 min. Then take 10 μL and mix it with the methylene blue staining solution (990 μL). After staining for 10 min, according to the principle that living cells can reduce the staining agent entering the cells and are not stained, count the number of living cells under a microscope using a hemocytometer, determine the number of living cells, and finally determine the cell survival rate according to the formula "Survival rate / % = Number of living cells / (Number of living cells + Number of dead cells) × 100%".

[0087] III. Test results

[0088] It was found that the survival rates of the recombinant Saccharomyces cerevisiae LKF-03 strain stored in a -20 °C environment for 1, 3, and 7 d were 65.2%, 27.9%, and 21.9% respectively. While the survival rates of the Saccharomyces cerevisiae LKF-01 strain stored in a -20 °C environment for 1, 3, and 7 d were only 50.8%, 17.3%, and 7.73% respectively, indicating that by transferring the recombinant plasmid containing the MET17 gene into Saccharomyces cerevisiae in the present invention, the obtained recombinant Saccharomyces cerevisiae has a significantly improved tolerance to freezing compared with Saccharomyces cerevisiae, ensuring its normal fermentation growth in a frozen environment and significantly improving the quality of frozen products (such as foods, cosmetics, drugs, etc.).

[0089] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for enhancing the freezing tolerance of Saccharomyces cerevisiae, characterized in that: First, a recombinant Saccharomyces cerevisiae is constructed, and then the recombinant Saccharomyces cerevisiae is induced to express; the Saccharomyces cerevisiae is Saccharomyces cerevisiae ( Saccharomyces cerevisiae )LKF-01 strain, deposited in Guangdong Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058; Among them, one or more of the following biological materials are used to construct recombinant Saccharomyces cerevisiae: ①: MET17 gene; the nucleotide sequence of the MET17 gene is shown in SEQ ID NO: 15; ②: Recombinant expression vector containing ①; ③: Expression cassette containing ①; ④: Recombinant expression vector containing ③.

2. The method according to claim 1, characterized in that The recombinant expression vector is S.cry-EGFP-KanMX.

3. A method for constructing a recombinant Saccharomyces cerevisiae, characterized in that: First, a recombinant plasmid containing the MET17 gene is constructed, and then transferred into Saccharomyces cerevisiae; the nucleotide sequence of the MET17 gene is shown in SEQ ID NO: 15; the Saccharomyces cerevisiae is the LKF-01 strain of Saccharomyces cerevisiae, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with a deposit number of GDMCCNo: 66058.

4. The recombinant Saccharomyces cerevisiae constructed by the method according to claim 3.

5. Use of the recombinant Saccharomyces cerevisiae according to claim 4 in preparing a product with high tolerance to freezing.

6. The use according to claim 5, characterized in that: The product is one or more of food, cosmetics, and medicines.

7. A recombinant Saccharomyces cerevisiae, characterized in that: The recombinant Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-03 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66060.

8. Use of MET17 gene or MET17 protein in enhancing the freezing tolerance of Saccharomyces cerevisiae, characterized in that: First, a recombinant plasmid containing the MET17 gene is constructed, and then transferred into Saccharomyces cerevisiae; the nucleotide sequence of the MET17 gene is shown in SEQ ID NO: 15; the amino acid sequence of the MET17 protein is shown in SEQ ID NO: 16; the Saccharomyces cerevisiae LKF-01 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with a deposit number of GDMCCNo: 66058.

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