CN116064266B - Recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and a construction method and application thereof, and belongs to the technical field of bioengineering. The invention clones fatty acid synthesis precursor to the salt-tolerant Saccharomyces cerevisiae (Zygosaccharomyces rouxii 3792), controls the key gene (ACSS) for synthesizing acetyl coenzyme A, and converts Saccharomyces cerevisiae (Saccharomyces cerevisiae CEN.PK2-1C) by connecting to a bidirectional expression plasmid pY15TEF-1 capable of being copied in Saccharomyces cerevisiae to obtain Saccharomyces cerevisiae genetic engineering bacteria with improved salt tolerance.

Description

Recombinant Saccharomyces cerevisiae with enhanced salt stress resistance and its construction method and application

Technical Field

The invention relates to the technical field of bioengineering, and in particular to a recombinant brewer's yeast with enhanced salt stress resistance, and a construction method and application thereof.

Background technique

Microorganisms are easily disturbed by various environmental factors during fermentation and growth, including exogenous and endogenous environments.

Zygosaccharomyces rouxii is a yeast that is resistant to high osmotic pressure. It can grow in materials with high sugar and salt content, and even under saturated salt conditions, its growth cannot be completely inhibited. Zygosaccharomyces rouxii is an important strain for brewing soy sauce and paste. It can give the product aroma components such as ethanol, esters, furfural, succinic acid, and furanone. Therefore, it is widely used in the production process of traditional high-salt fermented foods such as bean paste, soy sauce, and miso.

Saccharomyces cerevisiae is an important industrial model strain and is widely used in the production of alcoholic foods. However, unfavorable brewing environmental factors, such as salt stress, often make it difficult for Saccharomyces cerevisiae to maintain high-intensity fermentation production.

In view of this, the present invention is proposed.

Summary of the invention

The purpose of the present invention is to provide a recombinant Saccharomyces cerevisiae with enhanced salt stress resistance, and a construction method and application thereof. By overexpressing the acetyl-CoA synthetase gene (ACSS) in Saccharomyces cerevisiae, the fatty acid synthesis capacity can be improved, thereby enhancing the salt stress resistance of Saccharomyces cerevisiae.

To achieve the above object, the present invention constructs a recombinant Saccharomyces cerevisiae with enhanced salt stress resistance. The present invention first introduces a vector containing the acetyl-CoA synthetase gene of Zygosaccharomyces rouxii into Saccharomyces cerevisiae, and finds that the fatty acid synthesis ability of the recombinant bacteria introduced with the vector containing the acetyl-CoA synthetase gene is improved, thereby enhancing the salt stress tolerance of the recombinant bacteria.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a recombinant Saccharomyces cerevisiae with enhanced salt stress resistance, wherein the recombinant bacterium contains exogenous genes; the exogenous genes include acetyl-CoA synthetase genes.

In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant Saccharomyces cerevisiae, which comprises: connecting the acetyl-CoA synthetase gene to an expression plasmid, and then introducing the recombinant expression plasmid into Saccharomyces cerevisiae to obtain the recombinant Saccharomyces cerevisiae.

In a third aspect, the present invention provides the use of the above-mentioned recombinant Saccharomyces cerevisiae in the fields of fermentation and brewing and fermented food.

In a fourth aspect, the present invention provides a method for enhancing the salt stress resistance of Saccharomyces cerevisiae, which comprises using the above-mentioned recombinant Saccharomyces cerevisiae to overexpress the acetyl-CoA synthetase gene to control fatty acid synthesis.

The present invention has the following beneficial effects:

The present invention clones and expresses the ACSS gene of Saccharomyces rouxii and introduces it into Saccharomyces cerevisiae to induce the expression of acetyl-CoA synthetase gene, thereby enhancing the fatty acid synthesis ability and salt stress tolerance of Saccharomyces cerevisiae. The results show that after overexpressing ACSS, the total amount of fatty acids increased by 18.4% compared with the non-overexpressed strain, and the number of viable cells of the ACSS-overexpressing strain under salt stress conditions increased by 52.17%.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

FIG. 1 is the total amount of fatty acids in Example 1 and Comparative Example 1.

FIG. 2 is the statistical results of the number of viable bacteria in Example 1 and Comparative Example 1.

Detailed ways

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The invention provides a recombinant brewer's yeast with enhanced salt stress resistance. The recombinant yeast contains exogenous genes, and the exogenous genes include acetyl-CoA synthetase genes.

At present, acetyl-CoA is an important intermediate metabolite of energy metabolism and a pivotal substance in the metabolism of energy in the body. The three major nutrients, sugar, fat, and protein, converge into a common metabolic pathway through acetyl-CoA - the tricarboxylic acid cycle and oxidative phosphorylation. Through this pathway, they are completely oxidized to generate carbon dioxide and water, releasing energy for the synthesis of ATP. Acetyl-CoA is a precursor for the synthesis of energy substances such as fatty acids and ketone bodies, and is also a precursor for the synthesis of physiologically active substances such as cholesterol and its derivatives.

The present invention has found through creative research that acetyl-CoA not only plays a key role in the metabolism of energy substances, but also can regulate the tolerance of organisms to adverse environments. Based on this, the inventors of the present invention introduced a vector containing the acetyl-CoA synthetase gene of Saccharomyces rouxii into Saccharomyces cerevisiae and induced the synthesis of acetyl-CoA. The test results show that the fatty acid synthesis ability of the constructed recombinant bacteria is improved, thereby making it more capable of overcoming adverse environmental factors in fermentation production, especially salt stress environment.

In some embodiments, the acetyl-CoA synthetase gene is derived from Saccharomyces rouxii.

Saccharomyces rouxii is a yeast that is resistant to high osmotic pressure. It can grow in materials with high sugar and salt content, and even under saturated salt conditions, its growth cannot be completely inhibited. Saccharomyces rouxii is an important strain for brewing soy sauce and paste. It can give the product aroma components such as ethanol, esters, furfural, succinic acid, and furanone. Therefore, it is widely used in the production process of traditional high-salt fermented foods such as bean paste, soy sauce, and miso.

In some embodiments, the above-mentioned Z. rouxii is the Z. rouxii with a deposit number of CGMCC No. 3791, which was deposited on April 29, 2010 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

In some embodiments, the NCBI gene ID of the acetyl-CoA synthetase gene is ZYGR_0I00290.

In some embodiments, the nucleotide sequence of the acetyl-CoA synthetase gene is SEQ ID NO.1.

In some embodiments, the starting strain of recombinant Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C, disclosed in van Dijken JP et al., Enzyme Microb. Technol. 2000, 26:706-714, with a genotype of MATaura3-52 leu3-5,112trp1-289his3ΔMAL2-8 c SUC2, which was kindly donated by Jiangnan University.

In some embodiments, the expression plasmid of the recombinant Saccharomyces cerevisiae is a high-copy bidirectional expression plasmid.

In some embodiments, the above-mentioned expression plasmid includes pY15TEF-1 (Biovector Co., LTD), which is labeled with leucine LEU and was donated by Jiangnan University.

The present invention also provides a method for constructing the above-mentioned recombinant Saccharomyces cerevisiae, which comprises: connecting the acetyl-CoA synthetase gene to an expression plasmid, and then introducing the recombinant expression plasmid into Saccharomyces cerevisiae to obtain the recombinant Saccharomyces cerevisiae.

Specifically, the construction method includes the following steps:

(1) The target gene ACSS with two restriction sites, Hind III and Xho I, and protective bases at both ends was ligated to pY15TEF1 that had been digested with the same enzymes and transformed into Escherichia coli DH5a. The transformants obtained were verified by colony PCR, extracted plasmid restriction enzyme digestion verification, and sequencing to confirm that they were the recombinant plasmid pY15TEF1-ACSS.

(2) The constructed recombinant plasmid was transformed into Saccharomyces cerevisiae CEN.PK2-1C to obtain recombinant Saccharomyces cerevisiae with improved salt stress resistance.

The present invention also provides the application of the recombinant saccharomyces cerevisiae in the fields of fermentation and brewing and fermented food.

Specifically, the recombinant brewer's yeast constructed by the present invention can be applied to the production and fermentation of fermented soybeans, soy sauce, miso, etc. in the field of fermented foods.

The present invention also provides a method for enhancing the salt stress resistance of Saccharomyces cerevisiae, which comprises using the above-mentioned recombinant Saccharomyces cerevisiae to overexpress acetyl-CoA synthetase gene to control fatty acid synthesis.

In some embodiments, overexpression is to first construct a recombinant plasmid containing a gene encoding acetyl-CoA synthetase by combining a gene encoding acetyl-CoA synthetase with an expression plasmid, and then introduce the recombinant plasmid into Saccharomyces cerevisiae to induce the expression of acetyl-CoA synthetase.

In some embodiments, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C, whose genotype is MATaura3-52 leu3-5,112trp1-289 his3ΔMAL2-8 c SUC2.

In some embodiments, the acetyl-CoA synthetase gene is derived from Saccharomyces rouxii.

In some embodiments, the deposit number of Saccharomyces rouxii is CGMCC No.3791.

In some embodiments, the NCBI gene ID of the acetyl-CoA synthetase gene is ZYGR_0I00290.

In some embodiments, the nucleotide sequence of the acetyl-CoA synthetase gene is SEQ ID NO.1.

In some embodiments, the expression plasmid is a high copy bidirectional expression plasmid.

In some embodiments, the expression plasmid comprises pY15TEF-1.

The present invention will be further described below in conjunction with specific embodiments.

Example 1

This embodiment provides a recombinant Saccharomyces cerevisiae and a method for constructing the same.

The foreign gene in the recombinant Saccharomyces cerevisiae is the ACSS gene with the nucleotide sequence of SEQ ID NO. 1. The host is Saccharomyces cerevisiae CEN.PK2-1C; the vector is pY15TEF-1, all of which were donated by Jiangnan University.

The construction method is:

(1) Total RNA was extracted from Saccharomyces rouxii (Z.rouxii CGMCC No.3791), and the acetyl-CoA synthetase gene ACSS with two restriction sites, Xho I and Hind III, at both ends was obtained by PCR. The primers used to clone ACSS were:

F1:CCCTCGAGATGACGGTCAATTATGTATATGCAGG, SEQ ID NO.2;

R1:CCCAAGCTTCTACAATTTTACAGAATCAATCAAACGC, SEQ ID NO.3.

(2) Then, the plasmid pY15TEF1, which had been digested with the same enzyme, was ligated to the plasmid and transformed into Escherichia coli DH5α. The transformants were verified by colony PCR, and the extracted plasmid was verified by enzyme digestion and sequencing to confirm that it was the recombinant plasmid pY15TEF1-ACSS.

(3) The constructed recombinant plasmid was transformed into S. cerevisiae CEN.PK2-1C, the plasmid of the transformant was extracted, and the transformant was confirmed to be a positive transformant by enzyme digestion.

Comparative Example 1

The recombinant Saccharomyces cerevisiae constructed in this comparative example was as follows: the empty plasmid pY15TEF1 was transformed into Saccharomyces cerevisiae CEN.PK2-1C, the plasmid of the transformant was extracted, and the transformant was confirmed to be a positive transformant by enzyme digestion verification.

Experimental Example 1

This experimental example is for the determination of fatty acid content, and the specific steps are as follows:

(1) The strains of Example 1 and the comparative example, namely, recombinant Saccharomyces cerevisiae CEN.PK2-1C pY15TEF1-ACSS and S. cerevisiae CEN.PK2-1C pY15TEF1, which were stored in a glycerol storage solution at -80°C, were inoculated into a seed culture medium at an inoculum rate of 10% (V/V) and cultured at 30°C for 12 h until the mid-logarithmic phase.

The components of the seed culture medium include: 6.7 g/L yeast-free nitrogen source medium (YNB without (NH ₄ ) ₂ SO ₄ ), 20 g/L glucose, 0.02 g/L tryptophan Try, 0.02 g/L histidine His, and 0.02 g/L uracil Ura.

(2) Collect the cells to determine the fatty acid content. The method for fatty acid extraction and determination is published in Wu et al., J. Ind. Microbiol. Biotechnol. 2012, 39: 1031-1039. The results are shown in FIG1 .

As can be seen from Figure 1, after overexpression of ACSS, the total amount of fatty acids increased by 18.4% compared with the starting strain.

Experimental Example 2

This experimental example is a test of resistance under salt stress conditions. The specific steps are as follows:

(1) The strains of Example 1 and the comparative example, namely, recombinant Saccharomyces cerevisiae CEN.PK2-1C pY15TEF1-ACSS and S. cerevisiae CEN.PK2-1C pY15TEF1, which were stored in a glycerol storage solution at -80°C, were inoculated into a seed culture medium at an inoculum rate of 10% (V/V), and statically cultured at 30°C for 12 h until the mid-logarithmic phase to obtain a seed culture solution.

The components of the seed culture medium include: 6.7 g/L yeast-free nitrogen source medium (YNB without (NH ₄ ) ₂ SO ₄ ), 20 g/L glucose, 0.02 g/L tryptophan Try, 0.02 g/L histidine His, and 0.02 g/L uracil Ura.

(2) The seed culture solution was inoculated into the salt stress medium at an inoculum rate of 10% (V/V) and treated for 4 h.

The components of the salt stress medium include: 6.7 g/L yeast-free nitrogen source medium (YNB without (NH ₄ ) ₂ SO ₄ ), 20 g/L glucose, 0.02 g/L tryptophan Try, 0.02 g/L histidine His, 0.02 g/L uracil Ura, and 70.2 g/L sodium chloride NaCl.

(3) The culture medium after salt stress treatment was centrifuged at 8000 r/min (4°C) for 5 min, the bacteria were collected and resuspended in sterile water to control the initial biomass to 0.5 _OD600 /mL. After dilution 1000 times, 5 μL was spread on a solid plate culture medium and cultured at 30°C for 48 h. The number of colonies was calculated. The results are shown in Figure 2.

The components of the solid culture medium include: 6.7 g/L yeast-free nitrogen source medium (YNB without (NH ₄ ) ₂ SO ₄ ), 20 g/L glucose, 0.02 g/L tryptophan Try, 0.02 g/L histidine His, 0.02 g/L uracil Ura, and 20 g/L agar.

As can be seen from Figure 2, after overexpressing ACSS, the number of viable cells of the ACSS strain increased by 52.17% compared with the starting strain. This proves that by overexpressing the acetyl-CoA synthetase gene that controls fatty acid synthesis in Saccharomyces cerevisiae, the fatty acid synthesis capacity can be improved, thereby enhancing the salt stress tolerance of the recombinant bacteria.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (6)

1. A method of enhancing salt stress resistance of saccharomyces cerevisiae, comprising overexpressing an acetyl-coa synthase gene in saccharomyces cerevisiae.

2. The method for enhancing salt stress resistance of saccharomyces cerevisiae according to claim 1, wherein the over-expression is to construct a recombinant plasmid containing a gene encoding acetyl-coa synthase through a gene encoding acetyl-coa synthase and an expression plasmid, and then introduce the recombinant plasmid into saccharomyces cerevisiae to induce the expression of acetyl-coa synthase.

3. The method of enhancing salt stress resistance of saccharomyces cerevisiae according to claim 2, wherein said acetyl-coa synthase gene is derived from saccharomyces rouxii.

4. A method of enhancing salt stress resistance of saccharomyces cerevisiae according to claim 3, wherein the collection number of the saccharomyces cerevisiae is CGMCC No.3791.

5. The method for enhancing salt stress resistance of Saccharomyces cerevisiae according to claim 4, wherein NCBI gene ID of the acetyl-CoA synthetase gene is ZYGR _0I00290 and the nucleotide sequence of the acetyl-CoA synthetase gene is SEQ ID NO.1.

6. The method for enhancing salt stress resistance of Saccharomyces cerevisiae according to claim 5, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C and the genotype thereof is MATA ura3-52 leu3-5,112 trp1-289 his3ΔMAL2-8C SUC2.