Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of saccharomyces cerevisiae strain
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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
Active Publication Date: 2020-04-28
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology
[0005] The purpose of the present invention is to solve the problem that Saccharomyces cerevisiae does not synthesize ethyl b
Method used
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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 breeding of industrial microorganisms, in particular to a method for constructing a saccharomyces cerevisiae strain withhigh yield of ethyl butyrate and application of the saccharomyces cerevisiae strain. The method comprises the following steps: overexpressing an acetyl coenzyme A acyltransferase gene Erg10, a 3-hydroxybutyryl coenzyme A dehydrogenase gene Hbd, a 3-hydroxybutyryl coenzyme A dehydratase gene Crt, a trans-2-enoyl coenzyme A reductase gene Ter and an alcohol acyltransferase gene AAT in an original strain so as to obtain a yeast strain EST with the capability of producing ethyl butyrate. Compared with an original strain which does not produce the ethyl butyrate, the ethyl butyrate yield of the saccharomyces cerevisiae strain reaches 77.33+/-3.79 mg/L. A strain EDST is obtained after the Ter gene and the AAT gene are subjected to double-copy expression, and the yield of ethyl butyrate reaches 99.65+/-7.32 mg/L. Compared with the EST strain, the yield is increased by 28.9%, meanwhile, 40.93+/-3.18 mg/L of ethyl crotonate is generated, and an unexpected technical effect is achieved. The saccharomyces cerevisiae strain lays a theoretical basis for brewing Chinese liquor which is excellent in flavor and more beneficial to health, and has wide 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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