A high-yielding ethyl butyrate-producing Saccharomyces cerevisiae strain and its construction method and use
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A technology of Saccharomyces cerevisiae and ethyl butyrate, applied in the field of bioengineering, can solve the problem of not synthesizing ethyl butyrate and the like, and achieve the effects of quality improvement and stability, broad market prospects and important market value
Active Publication Date: 2021-08-03
TIANJIN UNIV OF SCI & TECH
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[0005] The purpose of the present invention is to solve the problem that Saccharomyces cerevisiae does not synthesize ethyl butyrate in wine production, and to provide a method for constructing ethyl butyrate-producing Saccharomyces cerevisiae strains
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Embodiment 1
[0072] Embodiment 1: Construction of producing ethyl butyrate Saccharomyces cerevisiae strain
[0073] The starting strain CICC32315 used in this example. The Escherichia coli DH5a was purchased from Takara Company. The YPD medium is a general complete medium, and the solid medium contains 2% (mass percentage) imported agar powder.
[0074] According to the gene sequences and integrated plasmid sequences in NCBI Genebank, the following primers were designed, as shown in Table 1.
[0075] Table 1 Primers
[0076]
[0077]
[0078] The PCR amplification system used in this example is shown in Table 2.
[0079] Table 2 PCR amplification system
[0080]
[0081] The main construction process of the strain is as follows:
[0082] (1) Construction of Yep352-PE / PH / PC / PT / PA plasmid
[0083] Using Yep352-P as the basic plasmid, construct recombinant plasmids carrying genes Erg10, Hbd, Crt, Ter, AAT, referred to as recombinant plasmids Yep352-PE, Yep352-PH, Yep352-PC, Yep...
Embodiment 2
[0110] Example 2: Corn raw material thick mash fermentation experiment of the starting strain and the modified strain
[0111] (1) Corn mash fermentation experiment of recombinant strains EST, EDT, EDS and EDST and parental strain (AY14-ɑ)
[0112]The parental strain AY14-ɑ and the recombinant strains EST, EDT, EDS and EDST were subjected to corn mash fermentation experiments at the same time. The fermentation process roadmap: corn flour→soaking→liquefaction→saccharification→cooling→inoculation→fermentation→steaming→determination index;
[0113] Pick a ring of yeast cells, put them into test tubes containing 5mL of primary seed medium, culture at 30°C for 24 hours, and inoculate 10% of the inoculum into a 150mL Erlenmeyer flask containing 45mL of secondary seed medium , static culture at 30°C for 16h to the late logarithmic phase, inoculate the fermentation medium with 10% inoculum amount, and statically ferment at 30°C. Weigh once every 12 hours, and when the two weight los...
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Abstract
The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a method for constructing a high-yield ethyl butyrate-producing yeast strain and its application. By overexpressing the acetyl-CoA acyltransferase gene Erg10, the 3-hydroxybutyryl-CoA dehydrogenase gene Hbd, the 3-hydroxybutyryl-CoA dehydratase gene Crt, and the trans-2-enoyl-CoA gene in the starting strain The reductase gene Ter and the alcohol acyltransferase gene AAT were used to obtain a yeast strain EST capable of producing ethyl butyrate. Compared with the starting strain that did not produce ethyl butyrate, its ethyl butyrate production reached 77.33 ±3.79mg / L. After two copies of Ter gene and AAT gene were expressed, strain EDST was obtained, and the output of ethyl butyrate reached 99.65 ± 7.32 mg / L, which was 28.9% higher than that of EST strain, and also produced 40.93 ± 3.18 mg Ethyl crotonate in 1 / L has achieved unexpected technical effects, and has laid a theoretical foundation for brewing a liquor with excellent flavor and is more beneficial to health, and has broad market prospects.
Description
Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yield ethyl butyrate-yielding Saccharomyces cerevisiae strain and its construction method and application. Background technique: [0002] The main components of Chinese liquor are water and ethanol. The proportion of the two in liquor is as high as 97%-98%, and the remaining flavor substances only account for 2%-3%. However, with the continuous development of flavor chemistry, it is found that these The flavor substances with very little content determine the aroma and characteristics of liquor. Among the many trace components, ester is the most important type of compound, which has a pleasant fruity aroma, and an appropriate amount of ester can increase the flavor of wine. For Chinese liquor, ethyl acetate, ethyl caproate, ethyl lactate and ethyl butyrate are the four main aroma components of Chinese liquor, which directly determine the quality of liq...
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