CN116064266A - Recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and construction method and application thereof - Google Patents
Recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and construction method and application thereof Download PDFInfo
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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
Technical Field
The invention relates to the technical field of bioengineering, in particular to recombinant saccharomyces cerevisiae with enhanced salt stress resistance, and a construction method and application thereof.
Background
Microorganisms are susceptible to a variety of environmental disturbances during fermentation and growth, including exogenous and endogenous environments.
The rupeste (Zygosaccharomyces rouxii) is a high osmotic pressure resistant yeast which can grow in materials with very high sugar and salt content and which cannot fully inhibit its growth even in saturated salt conditions. The rupeste is an important strain for brewing soy sauce and sauce, and can endow the products with aroma components such as ethanol, esters, furfural, succinic acid, furanone and the like. Therefore, it is widely used in the production process of conventional high-salt fermented foods such as bean paste, soy sauce, miso, etc.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) is an important industrial model strain and is widely used in the production of alcoholic foods. However, adverse brewing environmental factors, such as stress conditions such as salt stress, often make it difficult for Saccharomyces cerevisiae to maintain high-strength fermentation production.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide recombinant saccharomyces cerevisiae with enhanced salt stress resistance, a construction method and application thereof, wherein the fatty acid synthesis capacity can be improved by over-expressing an acetyl coenzyme A synthetase gene (ACSS) in the saccharomyces cerevisiae, so that the salt stress resistance of the saccharomyces cerevisiae is enhanced.
In order to achieve the aim, the invention constructs the recombinant saccharomyces cerevisiae with enhanced salt stress resistance. In the invention, a vector containing an acetyl-CoA synthetase gene of a rue yeast (Zygosaccharomyces rouxii) is firstly introduced into saccharomyces cerevisiae (Saccharomyces cerevisiae), and the fatty acid synthesis capability of recombinant bacteria introduced with the vector containing the acetyl-CoA synthetase gene is found to be improved, so that the salt stress tolerance capability of the recombinant bacteria is further enhanced.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a recombinant saccharomyces cerevisiae with enhanced salt stress resistance, wherein the recombinant saccharomyces cerevisiae contains exogenous genes; the foreign gene includes acetyl-CoA synthetase gene.
In a second aspect, the present invention provides a method for constructing the recombinant saccharomyces cerevisiae, comprising: and (3) connecting an acetyl-CoA synthetase gene to the expression plasmid, and then introducing the recombinant expression plasmid into saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae.
In a third aspect, the invention provides the use of the recombinant Saccharomyces cerevisiae described above in the field of fermentation brewing and fermented food.
In a fourth aspect, the present invention provides a method for enhancing salt stress resistance of Saccharomyces cerevisiae comprising over-expressing an acetyl-CoA synthetase gene using the recombinant Saccharomyces cerevisiae described above to control fatty acid synthesis.
The invention has the following beneficial effects:
the invention enhances the synthesis capability and salt stress tolerance capability of saccharomyces cerevisiae fatty acid by cloning and expressing the ACSS gene of the saccharomyces cerevisiae and introducing the ACSS gene into the saccharomyces cerevisiae to induce the expression of the acetyl-CoA synthetase gene. The result shows that after the ACSS is over-expressed, the total fatty acid amount is improved by 18.4 percent compared with that of a non-over-expressed strain, and the viable count of the over-expressed ACSS strain is improved by 52.17 percent under the condition of salt stress.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the total fatty acid amount of example 1 and comparative example 1.
FIG. 2 shows the results of statistics of the number of viable bacteria in example 1 and comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a recombinant saccharomyces cerevisiae with enhanced salt stress resistance, which contains exogenous genes; the foreign gene includes acetyl-CoA synthetase gene.
At present, acetyl-CoA is an important intermediate metabolite of energy substance metabolism, which is a pivotal substance in energy substance metabolism in vivo. The three nutrient substances of sugar, fat and protein are converged into a common metabolism path through acetyl coenzyme A, namely tricarboxylic acid circulation and oxidative phosphorylation, and carbon dioxide and water are thoroughly oxidized through the path, so that energy is released for synthesizing ATP. Acetyl-coa is a precursor for synthesizing energy substances such as fatty acids and ketones, and also for synthesizing physiologically active substances such as cholesterol and its derivatives.
The invention discovers that acetyl coenzyme A plays a key role in energy substance metabolism and can regulate the tolerance of organisms to adverse environments through creative researches. Based on the above, the inventors of the present invention introduced a vector containing an acetyl-CoA synthetase gene of Saccharomyces rouxii into Saccharomyces cerevisiae and induced the synthesis of acetyl-CoA, and the experimental results showed that the fatty acid synthesis ability of the constructed recombinant bacteria was improved, thereby enabling to overcome adverse environmental factors in fermentation production, particularly salt stress environment.
In some embodiments, the acetyl-coa synthetase gene is derived from saccharomyces rouxii.
The Russell microzyme is a microzyme with high osmotic pressure resistance, can grow in materials with high sugar content and high salt content, and can not completely inhibit the growth even under saturated salt condition. The rupeste is an important strain for brewing soy sauce and sauce, and can endow the products with aroma components such as ethanol, esters, furfural, succinic acid, furanone and the like. Therefore, it is widely used in the production process of conventional high-salt fermented foods such as bean paste, soy sauce, miso, etc.
In some embodiments, the foregoing rouxii is rouxii with a preservation number of CGMCC No.3791, a preservation time of 4 months and 29 days in 2010, a preservation place of the general microbiological center of the China Committee for culture Collection of microorganisms, and an address of the general microbiological center of the Beijing area, the Chaoyang area, the Beichen, no.1, and No. 3.
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 Di jken JP et al, enzyme microb.technology.2000, 26:706-714, genotype MATA ura3-52leu3-5,112trp1-289 his3 ΔMAL2-8C SUC2, benefit of university of Jiangnan.
In some embodiments, the expression plasmid of the recombinant saccharomyces cerevisiae described above is a high copy bi-directional expression plasmid.
In some embodiments, the expression plasmid described above comprises pY15TEF-1 (Biovector co., LTD) with a leucine LEU tag, offered by university of south of the river.
The invention also provides a construction method of the recombinant saccharomyces cerevisiae, which comprises the following steps: and (3) connecting an acetyl-CoA synthetase gene to the expression plasmid, and then introducing the recombinant expression plasmid into saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae.
Specifically, the construction method comprises the following steps:
(1) The target gene ACSS with two restriction sites of HindIII and XhoI and protective base at two ends is connected to pY15TEF1 which is subjected to the same restriction enzyme digestion to transform escherichia coli DH5a, and the obtained transformant is verified by colony PCR, extracted plasmid restriction enzyme digestion verification and sequencing to verify the recombinant plasmid pY15TEF1-ACSS.
(2) And (3) transforming the constructed recombinant plasmid into Saccharomyces cerevisiae CEN.PK2-1C to obtain the recombinant Saccharomyces cerevisiae with improved salt stress resistance.
The invention also provides application of the recombinant saccharomyces cerevisiae in the fields of fermentation brewing and fermented foods.
Specifically, the recombinant saccharomyces cerevisiae constructed by the invention can be applied to the production fermentation of bean paste, soy sauce, miso and the like in the field of fermented foods.
The invention also provides a method for enhancing the salt stress resistance of the saccharomyces cerevisiae, which comprises the step of utilizing the recombinant saccharomyces cerevisiae to overexpress an acetyl-CoA synthetase gene so as to control the synthesis of fatty acids.
In some embodiments, the overexpression is performed by constructing a recombinant plasmid containing a gene encoding acetyl-CoA synthetase from a gene encoding acetyl-CoA synthetase and an expression plasmid, and introducing the recombinant plasmid into Saccharomyces cerevisiae to induce expression of the acetyl-CoA synthetase.
In some embodiments, the Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C, which has a genotype of MATA ura3-52leu3-5,112trp1-289 his3ΔMAL2-8C SUC2.
In some embodiments, the acetyl-coa synthetase gene is derived from saccharomyces rouxii.
In some embodiments, the collection number of the 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 invention is further illustrated below in conjunction with specific examples.
Example 1
The embodiment provides a recombinant saccharomyces cerevisiae and a construction method thereof.
Wherein, the exogenous genes in the recombinant saccharomyces cerevisiae are: the nucleotide sequence is ACSS gene of SEQ ID NO.1. The host is: saccharomyces cerevisiae CEN.PK2-1C; the vector is pY15TEF-1, and is a benefit of Jiang Nada.
The construction method comprises the following steps:
(1) Extracting total RNA of the Ruhrymal microzyme (Z.rouxii CGMCC No. 3791), and obtaining an acetyl coenzyme A synthetase gene ACSS with two enzyme cutting sites of Xho I and Hind III at two ends by PCR. The primers used for cloning ACSS are:
F1:CCCTCGAGATGACGGTCAATTATGTATATGCAGG，SEQ IDNO.2；
R1:CCCAAGCTTCTACAATTTTACAGAATCAATCAAACGC，SEQ ID NO.3。
(2) Then connecting to pY15TEF1 plasmid subjected to same digestion, transforming escherichia coli DH5 alpha, obtaining transformant, and carrying out colony PCR verification, plasmid digestion verification and sequencing to confirm the recombinant plasmid pY15TEF1-ACSS.
(3) And transforming Saccharomyces cerevisiae (S.cerevisiae CEN.PK2-1C) with the constructed recombinant plasmid, extracting plasmids of the transformants, and performing enzyme digestion verification to confirm positive transformants.
Comparative example 1
The recombinant Saccharomyces cerevisiae constructed in this comparative example was: the empty plasmid pY15TEF1 was transformed into Saccharomyces cerevisiae (S.cerevisiae CEN.PK2-1C), and the plasmid of the transformant was extracted and verified by cleavage, and confirmed as a positive transformant.
Experimental example 1
The experimental example is for fatty acid content determination, and comprises the following specific steps:
(1) The strain of example 1 and comparative example, which was stored in glycerol stock at-80 ℃, was taken: recombinant Saccharomyces cerevisiae CEN.PK2-1C pY15TEF1-ACSS and S.cerevisiae CEN.PK2-1C pY15TEF1 were inoculated in seed medium at an inoculum size of 10% (V/V) and cultured at 30℃for 12h to mid-log phase.
The seed culture medium comprises the following components: 6.7g/L Yeast-freeNitrogen source Medium (YNB does not contain (NH) 4 ) 2 SO 4 ) Glucose 20g/L, tryptophan Try 0.02g/L, histidine His0.02g/L, uracil Ura 0.02g/L.
(2) The cells were collected for fatty acid content determination, and methods for fatty acid extraction and determination were published in Wu et al, j.ind.microbiol.biotechnol.2012, 39:1031-1039. The results are shown in FIG. 1.
As can be seen from FIG. 1, after overexpression of ACSS, the total fatty acid is increased by 18.4% compared to the starting strain.
Experimental example 2
The experimental example is the detection of the resistance under the condition of salt stress, and comprises the following specific steps:
(1) The strain of example 1 and comparative example, which was stored in glycerol stock at-80 ℃, was taken: recombinant Saccharomyces cerevisiae CEN.PK2-1C pY15TEF1-ACSS and S.cerevisiae CEN.PK2-1C pY15TEF1 are inoculated into a seed culture medium with an inoculum size of 10% (V/V) and are subjected to static culture at 30 ℃ for 12 hours to mid-log phase to obtain a seed culture solution.
The seed culture medium comprises the following components: 6.7g/L yeast-free nitrogen source medium (YNB does not contain (NH) 4 ) 2 SO 4 ) Glucose 20g/L, tryptophan Try 0.02g/L, histidine His0.02g/L, uracil Ura 0.02g/L.
(2) Seed culture broth was inoculated into salt stress medium at an inoculum size of 10% (V/V) for 4h.
The components of the salt stress culture medium comprise: 6.7g/L yeast-free nitrogen source medium (YNB does not contain (NH) 4 ) 2 SO 4 ) Glucose 20g/L, tryptophan Try 0.02g/L, histidine His0.02g/L, uracil Ura 0.02g/L, sodium chloride NaCl 70.2g/L.
(3) Centrifuging the culture solution subjected to salt stress at 8000r/min (4deg.C) for 5min, collecting thallus, suspending the thallus in sterile water, and controlling initial biomass to 0.5OD 600 After dilution by 1000 times, 5. Mu.L of the culture medium was applied to a solid plate medium, and the culture was allowed to stand at 30℃for 48 hours to calculate the colony count, and the result was shown in FIG. 2.
The solid medium comprises the following components: 6.7g/L yeast-free nitrogen sourceCulture medium (YNB does not contain (NH) 4 ) 2 SO 4 ) Glucose 20g/L, tryptophan Try 0.02g/L, histidine His0.02g/L, uracil Ura 0.02g/L, agar 20g/L.
As can be seen from FIG. 2, after the ACSS was overexpressed, the viable count of the ACSS strain was increased by 52.17% as compared to the starting strain. From this, it was demonstrated that the fatty acid synthesis ability can be improved by overexpressing an acetyl-CoA synthetase gene controlling fatty acid synthesis in Saccharomyces cerevisiae, thereby enhancing the salt stress tolerance of recombinant bacteria.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A recombinant saccharomyces cerevisiae with enhanced salt stress resistance, which is characterized in that the recombinant saccharomyces cerevisiae contains exogenous genes; the exogenous gene includes an acetyl-CoA synthetase gene.
2. The recombinant saccharomyces cerevisiae according to claim 1, wherein said acetyl-coa synthase gene is derived from saccharomyces rouxii;
preferably, the preservation number of the rupeste is CGMCC No.3791;
preferably, the NCBI gene ID of the acetyl-coa synthetase gene is zygr_0i00290;
preferably, the nucleotide sequence of the acetyl-CoA synthetase gene is SEQ ID NO.1.
3. The recombinant s.cerevisiae according to claim 2, wherein the starting strain of the recombinant s.cerevisiae is s.cerevisiae cen.pk2-1C, which has a genotype of MATa ura3-52leu3-5,112trp1-289 his3Δmal 2-8C sud 2.
4. The recombinant saccharomyces cerevisiae according to claim 3 wherein said recombinant saccharomyces cerevisiae contains high copy bidirectional expression plasmids;
preferably, the expression plasmid comprises pY15TEF-1.
5. The method for constructing a recombinant saccharomyces cerevisiae according to any of the claims 1-4, wherein said method for constructing comprises: and connecting the acetyl-CoA synthetase gene to the expression plasmid, and then introducing the recombinant expression plasmid into saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae.
6. Use of the recombinant saccharomyces cerevisiae according to any of claims 1-4 in the field of fermentation brewing and fermented food products.
7. A method of enhancing salt stress resistance of saccharomyces cerevisiae, comprising overexpressing an acetyl-coa synthase gene in saccharomyces cerevisiae.
8. The method for enhancing salt stress resistance of Saccharomyces cerevisiae according to claim 7, wherein the over-expression is to construct a recombinant plasmid containing a gene encoding acetyl-CoA synthetase by using a gene encoding acetyl-CoA synthetase and an expression plasmid, and then introduce the recombinant plasmid into Saccharomyces cerevisiae to induce expression of acetyl-CoA synthetase.
9. The method of enhancing salt stress resistance of saccharomyces cerevisiae according to claim 8, wherein said acetyl coa synthase gene is derived from saccharomyces rouxii;
preferably, the preservation number of the rupeste is CGMCC No.3791;
preferably, the NCBI gene ID of the acetyl-coa synthetase gene is zygr_0i00290;
preferably, the nucleotide sequence of the acetyl-CoA synthetase gene is SEQ ID NO.1.
10. The method of enhancing salt stress resistance of saccharomyces cerevisiae according to claim 9, wherein the saccharomyces cerevisiae is saccharomyces cerevisiae cen.pk2-1C with genotype MATa ura3-52leu3-5,112trp1-289 his3Δmal2-8C sud 2.
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