Application of MET14 gene or MET14 protein in improving freezing resistance of saccharomyces cerevisiae

Abstract

The invention provides an application of an MET14 gene or an MET14 protein in improving the freezing resistance of saccharomyces cerevisiae. It is found for the first time that through overexpression of the MET14 gene or the MET14 protein, the freezing resistance of the saccharomyces cerevisiae can be remarkably improved, the saccharomyces cerevisiae can normally ferment and grow in frozen products, the excellent physiological activity of the saccharomyces cerevisiae in the frozen products is fully exerted, and then the quality of the frozen products is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to the application of MET14 gene or MET14 protein in improving the frost resistance of brewer's yeast. Background Art

[0002] Brewer's yeast is widely used in frozen products such as frozen foods, frozen medicines, and frozen cosmetics. For example: (1) In the field of frozen foods, brewer's yeast can be directly used to ferment frozen dough to improve its gas production capacity, or brewer's yeast fermentation products (including carbon dioxide and alcohol, etc.) can be used to improve the flavor and texture of frozen foods; (2) In the field of frozen medicines, brewer's yeast, as a probiotic, can be used to maintain intestinal health and enhance immunity; (3) In the field of frozen cosmetics, brewer's yeast fermentation products (including free amino acids, small molecule peptides, vitamins, etc.) can be used for whitening, moisturizing, and delaying skin aging.

[0003] However, brewer's yeast has poor frost resistance and is easily damaged during the freezing or freeze-drying process, resulting in deterioration in the quality of frozen products. For example: (1) In the frozen food field, it may cause the fermentation performance of the dough to decline, affecting the volume, texture and taste of finished products such as bread and steamed buns; (2) In the frozen pharmaceutical field, it may cause the survival rate of brewer's yeast cells to decrease, or the beneficial ingredients that can be produced to decrease, thereby affecting the stability and efficacy of the drug; (3) In the frozen cosmetics field, it may cause the active ingredients that can be produced by yeast cells to decrease, thereby reducing the yield or efficacy of the cosmetics.

[0004] Therefore, it is urgent to find a method that can significantly improve the frost resistance of brewer's yeast, which is crucial to improving the quality of frozen products. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide an application of the MET14 gene or the MET14 protein in improving the frost resistance of brewer's yeast. By overexpressing the MET14 gene or the MET14 protein, the frost resistance of brewer's yeast is significantly improved, so that it can ferment and grow normally in frozen products, give full play to its excellent physiological activity in frozen products, and thus significantly improve the quality of frozen products.

[0006] The first objective of the present invention is to provide an application of a MET14 gene or a MET14 protein in improving the freezing resistance of Saccharomyces cerevisiae.

[0007] The second object of the present invention is to provide a method for improving the frost resistance of brewer's yeast.

[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 use of the above-mentioned recombinant Saccharomyces cerevisiae in the preparation of frozen products.

[0010] The fifth object of the present invention is to provide the use of the MET14 gene and / or a recombinant vector capable of expressing the MET14 gene in constructing a recombinant Saccharomyces cerevisiae with high frost resistance.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention first discovered that by overexpressing adenylate sulfate kinase MET14 gene (nucleotide sequence as shown in SEQ ID NO:9) or MET14 protein (amino acid sequence as shown in SEQ ID NO:10), the frost resistance of Saccharomyces cerevisiae can be significantly improved, so that it can ferment and grow normally in frozen products, give full play to its excellent physiological activity in frozen products, and thus significantly improve the quality of frozen products. Therefore, the application of MET14 gene or MET14 protein in improving the frost resistance of Saccharomyces cerevisiae should be within the protection scope of the present invention.

[0012] Preferably, the cerevisiae yeast is cerevisiae ( Saccharomyces cerevisiae ) LKF-01 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0013] Based on this, the present invention also provides a method for improving the frost resistance of brewer's yeast, specifically: after constructing the recombinant brewer's yeast, inducing expression; Among them, one or more of the following biological materials are used to construct recombinant Saccharomyces cerevisiae: (1) MET14 gene; (2) an expression cassette comprising (1); (3) A recombinant expression vector containing (1) and / or (2).

[0014] Preferably, the brewer's yeast is the brewer's yeast LKF-01 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with a deposit number of GDMCC No: 66058, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0015] Preferably, the recombinant expression vector is S.cry-EGFP-KanMX.

[0016] Preferably, the method specifically comprises: first constructing a recombinant plasmid containing the MET14 gene, and then transferring the recombinant plasmid into Saccharomyces cerevisiae to induce expression.

[0017] The recombinant brewer's yeast constructed by the above method has high frost resistance and is suitable for preparing frozen products. Therefore, the recombinant brewer's yeast constructed by the above method and the use of the recombinant brewer's yeast in preparing frozen products should be within the protection scope of the present invention.

[0018] Preferably, the recombinant Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-02 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66059, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0019] Preferably, the frozen product is one or more of frozen food, frozen medicine, and frozen cosmetics.

[0020] The present invention overexpresses the MET14 gene in Saccharomyces cerevisiae and finds that the recombinant Saccharomyces cerevisiae constructed has high frost resistance. Therefore, the use of the MET14 gene and / or a recombinant vector capable of expressing the MET14 gene in constructing a recombinant Saccharomyces cerevisiae with high frost resistance should also be within the scope of protection of the present invention.

[0021] Preferably, the recombinant Saccharomyces cerevisiae is the recombinant Saccharomyces cerevisiae LKF-02 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with a deposit number of GDMCC No: 66059, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0022] The present invention has the following beneficial effects: The present invention discovers for the first time that by overexpressing the MET14 gene or the MET14 protein, the frost resistance of brewer's yeast can be significantly improved, so that it can ferment and grow normally in frozen products, give full play to its excellent physiological activity in frozen products, and thus significantly improve the quality of frozen products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the agarose electrophoresis diagram of the plasmid S.cry-EGFP-KanMX in Example 1. Wherein, M is the marker band, and 1 is the PCR product band.

[0024] Figure 2 This is the agarose electrophoresis diagram of the plasmid S.cry-TEF1-MET14-EGFP-KanMX of Example 1. Wherein, M is the marker band, and 1 is the PCR product band.

[0025] Figure 3 It is the agarose electrophoresis diagram of Example 2. Wherein, M is the marker band, and 1 is the PCR product band.

[0026] Figure 4 This is the fluorescence microscopy image of Example 2. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples 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 art.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] Example 1 Construction of recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX S1. Using the genomic DNA of Saccharomyces cerevisiae LKF-01 strain (deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058) as a template, primers F1 / R1 were used to amplify the MET14 gene, primers F2 / R2 were used to amplify the promoter TEF1, primers F3 / R3 were used to amplify the upstream rDNA homologous sequence, and primers F4 / R4 were used to amplify the downstream rDNA homologous sequence.

[0030] The primers are shown in Table 1.

[0031] Table 1

[0032] The nucleotide sequence of the MET14 gene is shown in SEQ ID NO:9, and the amino acid sequence encoded by it is shown in SEQ ID NO:10.

[0033] SEQ ID NO:9: atggctactaatattacttggcatccaaatcttacttacgacgaacgcaaggcattgagaaaacaggacggttgtactatttggttaacaggtctaagtgcgtcaggtaaaagtacaatcgcctgtgcgctagaacagttactgctccaaaaaaacttgtctgcatatagattggatggtgacaacattcgttttggattgaacaaggatttgggtttctcagaaaaggacagaaatgaaaacattcgtagaattagcgaagtttctaagctatttgctgattcatgtgctatttcaatcacctcatttatctctccatacagagttgacagagatagagctcgtgaactacataaggaggctggtttgaagttcattgaaatatttgttgatgttccattagaagtcgctgagcaaagggaccctaagggtttatacaagaaagctagggagggtgtaatcaaggagtttacaggtatttctgccccatatgaagcgccaaaagctccagagctacatttgagaaccgaccagaagacggttgaagaatgtgctaccattatttatgagtacttaatcagtgaaaaaatcatccgtaagcatttgtaa; SEQ ID NO:10: MATNITWHPNLTYDERKALRKQDGCTIWLTGLSASGKSTIACALEQLLLQKNLSAYRLDGDNIRFGLNKDLGFSEKDRNENIRRISEVSKLFADSCAISITSFISPYRVDRDRARELHKEAGLKFIEIFVDVPLEVAEQRDPKGLYKKAREGVIKEFTGISAPYEAPKAPELHLRTDQKTVEECATIIYEYLISEKIIRKHL。

[0034] The nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO:11: ccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattccccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaatttttttttttgatttttttctctttcgatgacctcccattgatatttaagttattaaatggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa。

[0035] The nucleotide sequence of the upstream rDNA homologous sequence is shown in SEQ ID NO:12:

[0036] The nucleotide sequence of the downstream rDNA homologous sequence is shown in SEQ ID NO:13:

[0037] S2. Using plasmid pAUR123-EGFP as a template, primers F5 / R5 were used to amplify the EGFP+terminator ADH1 fragment sequence.

[0038] The primers are: Primer F5 (SEQ ID NO: 14): tctaacccgggtgatatcatggtgagcaagggcgagg; Primer R5 (SEQ ID NO: 15): ctgtcgattcgatactaacg.

[0039] The nucleotide sequence of the EGFP+terminator ADH1 fragment sequence is shown in SEQ ID NO: 16:

[0040] S3. Using plasmid pRCC-k as template, primers F6 / R6 were used to amplify the promoter TEF1+KanMX+terminator TEF1 fragment sequence, and primers F7 / R7 were used to amplify the Amp+ori fragment sequence.

[0041] The primers are shown in Table 2.

[0042] Table 2

[0043] The nucleotide sequence of the promoter TEF1+KanMX+terminator TEF1 fragment sequence is shown in SEQ ID NO: 21:

[0044] The nucleotide sequence of the Amp+ori fragment is shown in SEQ ID NO:22:

[0045] S4. The upstream rDNA homologous sequence, EGFP+terminator ADH1 fragment sequence, promoter TEF1+KanMX+terminator TEF1 fragment sequence, downstream rDNA homologous sequence and Amp+ori fragment sequence were recombined to obtain the plasmid S.cry-EGFP-KanMX.

[0046] S5. The plasmid S.cry-EGFP-KanMX obtained in S4 was transformed into Escherichia coli DH5α by heat shock method, and then spread on LB plate containing 100 μg / mL ampicillin (Amp). After culturing at 37 ℃ for 24 h, a single clone was picked and PCR verification was performed using the single clone as template and primer F3 (SEQ ID NO: 5) and primer R4 (SEQ ID NO: 8) as PCR verification primers. The PCR product was then subjected to agarose electrophoresis. The results are as follows: Figure 1 As shown in the figure, the band of the PCR product is above 4000 bp, indicating that the plasmid S.cry-EGFP-KanMX was successfully constructed.

[0047] S6. The plasmid S.cry-EGFP-KanMX obtained in S4 was double-digested with SmaI and EcoRV, and then recombined with the promoter TEF1 and MET14 genes to obtain the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX.

[0048] S7. The recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX obtained in S6 was transferred into Escherichia coli DH5α by heat shock method, and then spread on LB plate containing 100 μg / mL ampicillin (Amp). After culturing at 37 ℃ for 24 h, a single clone was picked and PCR verification was performed using the single clone as template and primer F2 (SEQ ID NO: 3) and primer R1 (SEQ ID NO: 2) as PCR verification primers. The PCR product was subjected to agarose electrophoresis. The results are as follows: Figure 2 As shown, the band of the PCR product is between 800 and 1200 bp, indicating that the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX was successfully constructed.

[0049] Example 2 Construction and screening of recombinant Saccharomyces cerevisiae S1. Using the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX obtained in Example 1 as a template, primer F3 (SEQ ID NO: 5) and primer R4 (SEQ ID NO: 8) as PCR specific primers, rDNA was amplified up-TEF1-MET14-EGFP-KanMX-rDNA down sequence.

[0050] S2. rDNA obtained from S1 up -TEF1-MET14-EGFP-KanMX-rDNA down The transformants were transformed into Saccharomyces cerevisiae LKF-01 strain by sequential electroporation and spread on yeast extract peptone glucose agar medium (YPD plate) containing 400 μg / mL Geneticin (G418) and 1 mol / L sorbitol. After culturing at 30°C for 72 h, the transformants were picked and inoculated into 1 mL yeast extract peptone glucose medium (YPD liquid medium), and cultured at 30°C and 180 rpm for 24 h to obtain culture solution.

[0051] S3. Take the transformant in S2, extract its genomic DNA, use the genomic DNA as a template, use primer F2 (SEQ ID NO: 3) and primer R5 (SEQ ID NO: 15) as PCR verification primers, perform PCR verification, and then perform agarose electrophoresis on the PCR product. The results are as follows: Figure 3 As shown. It can be seen that the band of the PCR product is around 2000 bp, and sequencing confirmed that it contains the nucleotide sequence shown in SEQ ID NO: 9, indicating that the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX has been successfully transferred into the Saccharomyces cerevisiae LKF-01 strain, and the recombinant Saccharomyces cerevisiae LKF-02 strain (deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number of GDMCC No: 66059) has been successfully constructed.

[0052] S4. Take the culture medium obtained in S2 and observe it under a fluorescence microscope (excitation wavelength of 465-495 nm, emission filter wavelength of 512-558 nm, 10x objective lens). The results are as follows: Figure 4 As shown, the fluorescence microscope image shows green fluorescence, and the EGFP in the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX is a green fluorescent protein, and it is fused with the MET14 protein, indicating that the overexpression of the MET14 protein in the recombinant Saccharomyces cerevisiae LKF-02 strain has been successfully completed.

[0053] Example 3 Freeze resistance test of recombinant Saccharomyces cerevisiae The cerevisiae Saccharomyces cerevisiae LKF-01 strain and the recombinant cerevisiae Saccharomyces cerevisiae LKF-02 strain obtained in Example 2 were inoculated into yeast extract peptone glucose medium (YPD liquid medium) containing 200 mg / mL Geneticin (G418), respectively, and cultured at 30°C and 180 rpm for 24 h. Then, 1 mL of the culture solution was dispensed into each tube, and the tubes were centrifuged at 8000 rpm for 5 min, and the supernatant was removed.

[0054] The precipitated bacterial cells obtained by centrifugation were placed in a -20 ℃ refrigerator for 1, 3, and 7 days, respectively, and then taken out, thawed at room temperature (25 ℃), and then 1 mL PBS was added to resuspend the bacterial cells. After 30 minutes, 10 μL was taken and mixed with methylene blue staining solution (990 μL). After staining for 10 minutes, according to the principle that living cells can reduce the dye entering the cells without being stained, the number of living cells was counted under a microscope using a hemocytometer to determine the number of living cells. Finally, the cell survival rate was determined according to the formula "survival rate / %=number of living cells / (number of living cells + number of dead cells)×100%". The preparation method of methylene blue staining solution is as follows: 0.025 g of methylene blue, 0.042 g of potassium chloride, 0.048 g of calcium chloride hexahydrate, 0.02 g of sodium bicarbonate, and 1.0 g of glucose were mixed, and then sterile saline was added to make up to 100 mL.

[0055] The results showed that the survival rates of the Saccharomyces cerevisiae LKF-01 strain stored in a -20 ℃ environment for 1, 3, and 7 days were 50.8%, 17.3%, and 7.73%, respectively, while the survival rates of the recombinant Saccharomyces cerevisiae LKF-02 strain stored in a -20 ℃ environment for 1, 3, and 7 days were 72.0%, 50.6%, and 23.3%, respectively. It can be seen that the present invention can significantly improve the survival rate of Saccharomyces cerevisiae in a frozen environment by transferring the recombinant plasmid containing the MET14 gene into Saccharomyces cerevisiae, that is, the overexpression of the MET14 gene in Saccharomyces cerevisiae can significantly improve the frost resistance of Saccharomyces cerevisiae, so that Saccharomyces cerevisiae can ferment and grow normally in frozen products, give full play to its excellent physiological activity in frozen products, and thus significantly improve the quality of frozen products.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of MET14 gene or MET14 protein in improving the frost resistance of Saccharomyces cerevisiae, characterized in that: The nucleotide sequence of the MET14 gene is shown in SEQ ID NO:9, and the amino acid sequence of the MET14 protein is shown in SEQ ID NO:10; the brewer's yeast is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-01 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66058.

2. A method for improving the frost resistance of brewer's yeast, characterized in that: After constructing the recombinant Saccharomyces cerevisiae, the expression is induced; the Saccharomyces cerevisiae is the Saccharomyces cerevisiae LKF-01 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with a deposit number of GDMCC No: 66058; Among them, one or more of the following biological materials are used to construct recombinant Saccharomyces cerevisiae: (1) MET14 gene, the nucleotide sequence of the MET14 gene is shown in SEQ ID NO:9; (2) an expression cassette comprising (1); (3) A recombinant expression vector containing (1) and / or (2).

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

4. The method according to claim 2, characterized in that: First, a recombinant plasmid containing the MET14 gene is constructed, and then the recombinant plasmid is transferred into Saccharomyces cerevisiae for inducing expression.

5. The recombinant Saccharomyces cerevisiae constructed by the method according to any one of claims 2 to 4.

6. Use of the recombinant Saccharomyces cerevisiae according to claim 5 in preparing frozen products.

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

8. Use of the MET14 gene and / or a recombinant vector capable of expressing the MET14 gene in constructing a recombinant Saccharomyces cerevisiae with high frost resistance, characterized in that: The nucleotide sequence of the MET14 gene is shown in SEQ ID NO: 9, and the recombinant Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) LKF-02 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 26, 2025, with the deposit number GDMCC No: 66059.

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