Application of MET14 gene or MET14 protein in enhancing the cold resistance of Saccharomyces cerevisiae

Abstract

The present invention provides the use of the MET14 gene or MET14 protein to enhance the frost tolerance of Saccharomyces cerevisiae. This invention is the first to discover that overexpressing the MET14 gene or MET14 protein can significantly enhance the frost tolerance of Saccharomyces cerevisiae, enabling normal fermentation and growth in frozen products, fully unleashing its excellent physiological activity in frozen products, and significantly improving the quality of frozen products.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology and more specifically relates to the use of a MET14 gene or a MET14 protein in improving the frost resistance of Saccharomyces cerevisiae. 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 poor quality of frozen products. For example: (1) in the frozen food field, it may lead to a decrease in the fermentation performance of the dough, affecting the volume, texture and taste of finished products such as bread and steamed buns; (2) in the frozen pharmaceutical field, it may lead to a decrease in the survival rate of brewer's yeast cells, or a decrease in the beneficial ingredients that can be produced, thereby affecting the stability and efficacy of the drugs; (3) in the frozen cosmetics field, it may lead to a decrease in the active ingredients that can be produced by yeast cells, thereby reducing the yield or efficacy of the cosmetics.

[0004] Therefore, there is an urgent need to find a method that can significantly improve the freeze resistance of brewer's yeast, which is crucial for improving the quality of frozen products. Summary of the Invention

[0005] In response to the deficiencies of the existing technology, the present invention aims to provide an application of the MET14 gene or MET14 protein in improving the frost resistance of brewer's yeast. By overexpressing the MET14 gene or MET14 protein, the frost resistance of brewer's yeast is significantly improved, enabling it to ferment and grow normally in frozen products, fully exerting its excellent physiological activity in frozen products, and thereby significantly improving the quality of frozen products.

[0006] The first object 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] A 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 recombinant Saccharomyces cerevisiae in preparing frozen products.

[0010] A fifth object of the present invention is to provide the use of a 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:

[0012] The present invention discovers for the first time that overexpressing the adenylate sulfate kinase (ASD) MET14 gene (nucleotide sequence shown in SEQ ID NO:9) or MET14 protein (amino acid sequence shown in SEQ ID NO:10) can significantly enhance the frost tolerance of Saccharomyces cerevisiae, enabling normal fermentation and growth in frozen products, fully exerting its excellent physiological activity in frozen products, and significantly improving the quality of frozen products. Therefore, the use of the MET14 gene or MET14 protein to enhance the frost tolerance of Saccharomyces cerevisiae falls within the scope of protection of the present invention.

[0013] 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.

[0014] Based on this, the present invention also provides a method for improving the frost resistance of Saccharomyces cerevisiae, specifically comprising: constructing a recombinant Saccharomyces cerevisiae and then inducing expression;

[0015] Among them, one or more of the following biological materials are used to construct recombinant Saccharomyces cerevisiae:

[0016] (1) MET14 gene;

[0017] (2) an expression cassette containing (1);

[0018] (3) A recombinant expression vector containing (1) and / or (2).

[0019] 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.

[0020] Preferably, the recombinant expression vector is S.cry-EGFP-KanMX.

[0021] Preferably, the method specifically comprises: first constructing a recombinant plasmid containing the MET14 gene, and then transferring the recombinant plasmid into Saccharomyces cerevisiae for inducing expression.

[0022] The recombinant Saccharomyces cerevisiae constructed by the above method has high frost resistance and is suitable for preparing frozen products. Therefore, the recombinant Saccharomyces cerevisiae constructed by the above method and the use of the recombinant Saccharomyces cerevisiae in preparing frozen products should be within the scope of protection of the present invention.

[0023] 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.

[0024] Preferably, the frozen product is one or more of frozen food, frozen medicine, and frozen cosmetics.

[0025] 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.

[0026] Preferably, the recombinant cerevisiae yeast is the recombinant cerevisiae yeast LKF-02 strain, which was deposited in the 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.

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

[0028] The present invention discovered for the first time that by overexpressing the MET14 gene or MET14 protein, the frost resistance of brewer's yeast can be significantly improved, enabling it to ferment and grow normally in frozen products, fully exerting its excellent physiological activity in frozen products, and thereby significantly improving the quality of frozen products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 2 This is the agarose electrophoresis diagram of the plasmid S.cry-TEF1-MET14-EGFP-KanMX in Example 1. Wherein, M is the marker band and 1 is the PCR product band.

[0031] Figure 3 This is the agarose electrophoresis diagram of Example 2, where M is the marker band and 1 is the PCR product band.

[0032] Figure 4 This is a fluorescence microscope image of Example 2. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0035] Example 1 Construction of recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX

[0036] S1. Using genomic DNA of Saccharomyces cerevisiae strain LKF-01 (deposited with Guangdong Provincial Microbial Culture Collection on March 26, 2025, with the deposit number GDMCC No: 66058) as a template, the MET14 gene was amplified with primers F1 / R1, the promoter TEF1 was amplified with primers F2 / R2, the upstream rDNA homologous sequence was amplified with primers F3 / R3, and the downstream rDNA homologous sequence was amplified with primers F4 / R4.

[0037] The primers are shown in Table 1.

[0038] Table 1

[0039]

[0040] 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.

[0041] SEQ ID NO:9:

[0042] atggctactaatattacttggcatccaaatcttacttacgacgaacgcaaggcattgagaaaacaggacggttgtactatttggttaacaggtctaagtgcgtcaggtaaaagtacaatcgcctgtgcgctagaacagttactgctccaaaaaaacttgtctgcatatagattggatggtgacaacattcgttttggattgaacaaggatttgggtttctcagaaaaggacagaaatgaaaacattcgtagaattagcgaagtttctaagctatttgctgattcatgtgctatttcaatcacctcatttatctctccatacagagttgacagagatagagctcgtgaactacataaggaggctggtttgaagttcattgaaatatttgttgatgttccattagaagtcgctgagcaaagggaccctaagggtttatacaagaaagctagggagggtgtaatcaaggagtttacaggtatttctgccccatatgaagcgccaaaagctccagagctacatttgagaaccgaccagaagacggttgaagaatgtgctaccattatttatgagtacttaatcagtgaaaaaatcatccgtaagcatttgtaa;

[0043] SEQ ID NO:10:

[0044] MATNITWHPNLTYDERKALRKQDGCTIWLTGLSASGKSTIACALEQLLLQKNLSAYRLDGDNIRFGLNKDLGFSEKDRNENIRRISEVSKLFADSCAISITSFISPYRVDRDRARELHKEAGLKFIEIFVDVPLEVAEQRDPKGLYKKAREGVIKEFTGISAPYEAPKAPELHLRTDQKTVEECATIIYEYLISEKIIRKHL。

[0045] The nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO:11:

[0046] ccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattccccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaatttttttttttgatttttttctctttcgatgacctcccattgatatttaagttattaaatggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa。

[0047] The nucleotide sequence of the upstream rDNA homologous sequence is shown in SEQ ID NO:12:

[0048]

[0049] The nucleotide sequence of the downstream rDNA homologous sequence is shown in SEQ ID NO: 13:

[0050]

[0051] S2. Using plasmid pAUR123-EGFP as a template, primers F5 / R5 were used to amplify the EGFP+terminator ADH1 fragment sequence.

[0052] Among them, the primers are:

[0053] Primer F5 (SEQ ID NO: 14): tctaacccgggtgatatcatggtgagcaagggcgagg;

[0054] Primer R5 (SEQ ID NO: 15): ctgtcgattcgatactaacg.

[0055] The nucleotide sequence of the EGFP+terminator ADH1 fragment sequence is shown in SEQ ID NO: 16:

[0056]

[0057] S3. Using plasmid pRCC-k as a 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.

[0058] The primers are shown in Table 2.

[0059] Table 2

[0060]

[0061] The nucleotide sequence of the promoter TEF1+KanMX+terminator TEF1 fragment sequence is shown in SEQ ID NO: 21:

[0062]

[0063] The nucleotide sequence of the Amp+ori fragment is shown in SEQ ID NO: 22:

[0064]

[0065] S4. Recombining 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 to obtain the plasmid S.cry-EGFP-KanMX.

[0066] S5. Heat-shock the plasmid S.cry-EGFP-KanMX obtained in S4 into Escherichia coli DH5α. Plate the plasmid onto an LB plate containing 100 μg / mL ampicillin (Amp). Incubate at 37°C for 24 h. Isolate a single clone and perform PCR verification using the clone as a template and primers F3 (SEQ ID NO: 5) and R4 (SEQ ID NO: 8). PCR products were then subjected to agarose gel electrophoresis. The results are shown in Figure 2. Figure 1 As shown in the figure, the PCR product band is above 4000 bp, indicating that the plasmid S.cry-EGFP-KanMX was successfully constructed.

[0067] 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.

[0068] S7. The recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX obtained in S6 was transformed into Escherichia coli DH5α by heat shock. The plasmid was then plated on LB plates containing 100 μg / mL ampicillin (Amp). After incubation at 37°C for 24 h, a single clone was selected. PCR verification was performed using the single clone as a template and primers F2 (SEQ ID NO: 3) and R1 (SEQ ID NO: 2) as PCR verification primers. The PCR products were then subjected to agarose electrophoresis. The results were as follows: Figure 2 As shown in Figure 3, the PCR product bands ranged from 800 to 1200 bp, indicating that the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX was successfully constructed.

[0069] Example 2 Construction and screening of recombinant Saccharomyces cerevisiae

[0070] S1. Using the recombinant plasmid S.cry-TEF1-MET14-EGFP-KanMX obtained in Example 1 as a template, primers F3 (SEQ ID NO: 5) and R4 (SEQ ID NO: 8) were used as PCR specific primers to amplify rDNA. up-TEF1-MET14-EGFP-KanMX-rDNA down sequence.

[0071] S2. rDNA obtained from S1 up -TEF1-MET14-EGFP-KanMX-rDNA down The transformants were transformed into the Saccharomyces cerevisiae LKF-01 strain by sequential electroporation and plated on yeast extract peptone dextrose agar medium (YPD plates) containing 400 μg / mL Geneticin (G418) and 1 mol / L sorbitol. After incubation at 30°C for 72 h, transformants were picked and inoculated into 1 mL of yeast extract peptone dextrose medium (YPD liquid medium). The culture was shaken at 30°C and 180 rpm for 24 h to obtain the culture medium.

[0072] S3. Take the transformant from S2 and extract its genomic DNA. Use the genomic DNA as a template and primers F2 (SEQ ID NO: 3) and R5 (SEQ ID NO: 15) as PCR verification primers to perform PCR verification. Then, perform agarose electrophoresis on the PCR product. The results are as follows: Figure 3 As shown, the PCR product band 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 transformed into the Saccharomyces cerevisiae LKF-01 strain, and the recombinant Saccharomyces cerevisiae LKF-02 strain (deposited with the Guangdong Provincial Center for Microbiological Culture Collection on March 26, 2025, with the deposit number GDMCC No: 66059) has been successfully constructed.

[0073] S4. Take the culture medium obtained in S2 and observe it under a fluorescence microscope (excitation wavelength 465-495 nm, emission filter wavelength 512-558 nm, 10x objective lens). The results are as follows: Figure 4 As shown in the fluorescence microscopy image, green fluorescence is observed, 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.

[0074] Example 3 Freeze-tolerance test of recombinant Saccharomyces cerevisiae

[0075] The Saccharomyces cerevisiae LKF-01 strain and the recombinant Saccharomyces cerevisiae LKF-02 strain obtained in Example 2 were respectively inoculated into yeast extract peptone dextrose medium (YPD liquid medium) containing 200 mg / mL Geneticin (G418). After shaking culture at 30°C and 180 rpm for 24 h, 1 mL of the culture medium was dispensed into each tube, and the culture was centrifuged at 8000 rpm for 5 min, and the supernatant was removed.

[0076] The bacterial pellet obtained by centrifugation was stored in a -20°C freezer for 1, 3, and 7 days, then removed, thawed at room temperature (25°C), and resuspended in 1 mL of PBS. After 30 minutes, 10 μL was mixed with 990 μL of methylene blue staining solution. After staining for 10 minutes, the number of viable cells was determined using a hemocytometer under a microscope, based on the principle that viable cells can reduce the dye that has entered the cells without being stained. Finally, the cell viability was determined using the formula "viability / % = number of viable cells / (number of viable cells + number of dead cells) × 100%." The methylene blue staining solution was prepared by mixing 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, and then diluting to 100 mL with sterile saline.

[0077] The results showed that the survival rates of the Saccharomyces cerevisiae LKF-01 strain after storage at -20°C for 1, 3, and 7 days were 50.8%, 17.3%, and 7.73%, respectively. The survival rates of the recombinant Saccharomyces cerevisiae LKF-02 strain after storage at -20°C for 1, 3, and 7 days were 72.0%, 50.6%, and 23.3%, respectively. This indicates that the transfer of the recombinant plasmid containing the MET14 gene into Saccharomyces cerevisiae in the present invention significantly improved the survival rate of Saccharomyces cerevisiae in a frozen environment. Specifically, overexpression of the MET14 gene in Saccharomyces cerevisiae significantly enhanced the frost tolerance of Saccharomyces cerevisiae, enabling the normal fermentation and growth of Saccharomyces cerevisiae in frozen products, fully exerting its excellent physiological activity in frozen products, and thus significantly improving the quality of frozen products.

[0078] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of the MET14 gene or MET14 protein in improving the frost resistance of Saccharomyces cerevisiae, characterized in that: The frost resistance of Saccharomyces cerevisiae is improved by overexpressing the MET14 gene or the MET14 protein; 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 Saccharomyces cerevisiae 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 Saccharomyces cerevisiae LKF-01 strain, characterized in that: The freezing resistance of Saccharomyces cerevisiae is improved by overexpressing the MET14 gene or MET14 protein; after constructing the recombinant Saccharomyces cerevisiae, the expression is induced; the Saccharomyces cerevisiae LKF-01 strain was deposited in the Guangdong Provincial Microbiological Culture Collection 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) MET14 gene, the nucleotide sequence of the MET14 gene is shown in SEQ ID NO: 9; (2) an expression cassette containing (1); (3) A recombinant expression vector containing (1) and / or (2).

3. 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 induced expression.

4. The recombinant Saccharomyces cerevisiae constructed by the method according to any one of claims 2 to 3.

5. Use of the recombinant Saccharomyces cerevisiae according to claim 4 in preparing frozen products.

6. The application according to claim 5, characterized in that The frozen product is frozen food or frozen medicine.

7. 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 freezing resistance of Saccharomyces cerevisiae is improved by overexpressing the MET14 gene or the MET14 protein; 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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