Saccharomyces cerevisiae highly producing C6-C10 ethyl esters and construction method and purpose of saccharomyces cerevisiae
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A C6-C10, Saccharomyces cerevisiae technology, applied in the breeding of industrial microorganisms, C6-C10 ethyl ester-producing Saccharomyces cerevisiae and its construction, can solve the problems of affecting the yield of raw materials, low efficiency, high cost, etc.
Active Publication Date: 2020-02-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology
On the one hand, these natural ester-producing yeasts and microorganisms that provide ester precursors do not have the ability to produce C6-C10 ethyl esters
On the other hand, the existence of wild flora seriously affects the yield of raw materials, making the alcohol fermentation efficiency less than one-third of that of Saccharomyces cerevisiae, resulting in high grain consumption, long production cycle, low efficiency and high cost of high-grade wine
[0004] In recent years, there have been many reports on ester production by non-Saccharomyces cerevisiae. These studies are either based on the presence of caproic acid, caprylic acid and capric acid, or mixed fermentation with Saccharomyces cerevisiae as the main body. C10 ethyl ester research reports, there is no research on the metabolic pathway of Saccharomyces cerevisiae to produce C6-C10 ethyl ester
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Embodiment 1
[0102] Example 1: Construction of high-yield C6-C10 ethyl ester Saccharomyces cerevisiae strain
[0103]The starting strain used in this example is Saccharomyces cerevisiae CA in Angelia yeast, and the strain preservation number is CGMCCNo.18670. The Escherichia coli DH5a was purchased from Takara Company. The YPD medium is a general complete medium, and the solid medium contains 2% imported agar powder.
[0104] The main construction process of the strain is as follows:
[0105] (1) Enhancing the synthesis of acetyl-CoA to produce C6-C10 ethyl ester Saccharomyces cerevisiae strain
[0106] Using the genome of yeast strain CA as a template and Gal80 as an integration site, PCR amplification with a primer pair of GA-U (SEQ ID NO:9) and GA-D (SEQ ID NO:10) yielded a 549bp TEF1 with a promoter P The upper homology arm GA segment of the homology region; use the primer pair GB-U (SEQ ID NO: 11) and GB-D (SEQ ID NO: 12) PCR amplification to obtain a size of 502bp with the screeni...
[0149] 1) The specific fermentation process is: corn flour→soaking→liquefaction→saccharification→cooling→inoculation→fermentation→steaming wine→determining indicators;
[0150] 2) Process conditions
[0151] Soaking conditions: 60-70°C, soaking for 20 minutes; liquefaction conditions: 85-90°C, adding high-temperature-resistant α-amylase, liquefying for 90 minutes; saccharification conditions: 55-60°C, adding glucoamylase, saccharifying for 20 hours;
[0152] 3) Ingredients: 1500g corn flour, 4500mL water, let stand for 20min, high temperature resistant α-amylase 2×10 4 U / mL, 0.9ml, glucoamylase 1×10 5 U / mL, 3mL.
[0153] 4) Culture medium configuration
[0154] Primary seed medium: 8°Brix corn hydrolyzate, add 0.5% yeast extract powder, aliquot 5mL into test tubes, boil for 10min to sterilize.
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Abstract
The invention discloses saccharomyces cerevisiae highly producing C6-C10 ethyl esters and a construction method and purpose of the saccharomyces cerevisiae, and belongs to the technical field of bioengineering. Through overexpressing acetaldehyde dehydrogenase ALD6 and acetyl-CoA synthase ASC1 in an original strain, synthesis of acetyl-CoA is strengthened, through overexpressing acetyl-CoA carboxylase ACC1**, malonyl CoA is strengthened, through overexpressing fatty acid synthetase FAS1 and fatty acid synthetase FAS2, medium-chain acyl-CoA is strengthened, more metabolism flows flow to medium-chain acyl groups CoA, alcohol acyl-transferase genes SAAT in strawberries are further exogenously introduced, and a reformed strain C-ald6acs1A*F1F2S is obtained. Under the condition of fermentation,the yield of ethyl caproate is 7.53mg/L which is 2.72 times of that of an original strain C-ald6acs1A*F1F2, and 26.89 times of that (only 0.27mg/L) of an original strain CA, the yield of ethyl caprylate is 13.65mg/L which is 9.11 times of that of the original strain CA, the yield of ethyl decanoate is 13.89mg/L which is 7.27 times of that of the original strain, and the saccharomyces cerevisiae has potential application prospects in improving flavor of wine and improving the quality of the wine.
Description
Technical field: [0001] The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a C6-C10 ethyl ester producing Saccharomyces cerevisiae and its construction method and application. Background technique: [0002] Ester aroma substances are the main flavor substances in wine. The higher ester content not only endows it with an important ester aroma, but also can effectively expand and relax nerves and reduce the side effects caused by drinking. C6-C10 ethyl ester refers to ethyl caproate, ethyl caprylate and ethyl caprate. C6-C10 ethyl esters are the important aroma substances of all aroma-type Chinese liquors, especially the characteristic aroma substances of Luzhou-flavor liquors and wines. Ethyl caproate, ethyl caprylate, and ethyl caprate had pineapple, apple, and fatty acid odors, respectively. Because Saccharomyces cerevisiae lacks the corresponding acyl-CoA and lacks specific alcohol acy...
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