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Construction method of over-expressed aromatic amino acid transaminase II Aro9 in beer yeast

An aromatic amino acid and brewer's yeast technology, applied in the field of genetic engineering, can solve problems such as unpleasant bitterness, achieve the effects of optimizing strains, increasing the original copy number, and increasing the number of genes

Pending Publication Date: 2020-09-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Higher alcohol, also known as miscellaneous alcohol, refers to alcohols containing more than 3 carbon atoms. An appropriate amount of higher alcohol can make the taste of beer soft and harmonious, and the wine body is round and mellow. However, if the content of higher alcohol in beer is too high, it will have an unpleasant taste. bitterness

Method used

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  • Construction method of over-expressed aromatic amino acid transaminase II Aro9 in beer yeast
  • Construction method of over-expressed aromatic amino acid transaminase II Aro9 in beer yeast
  • Construction method of over-expressed aromatic amino acid transaminase II Aro9 in beer yeast

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Experimental program
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Effect test

Embodiment 1

[0022] Obtaining of the target gene of embodiment 1

[0023] (1) Take the yeast suspension stored in the glycerol tube out of the -80°C refrigerator and place it in an ice-water mixture (or 4°C refrigerator) to thaw until the yeast suspension completely melts. Mix the yeast suspension evenly with a pipette, pipette 100 μL and spread it on the YPD plate, seal the plate with parafilm, and place it upside down in a 25°C incubator for cultivation. Cultivate for about 2-3 days, and after the single yeast colony grows to 2-3mm, place it in a refrigerator at 4°C for later use.

[0024] (2) Use an inoculation loop to pick a single colony of fresh yeast on a tightly streaked YPD plate, and culture it statically until the bacteria grow out. Scrape the bacteria from the plate, add EP tube containing 30 μL of SDS solution, and vortex Spin the mixer to mix well, put it in a 95°C water bath for 5 minutes, then place it on ice for 5 minutes, repeat three times, then centrifuge to absorb 20 ...

Embodiment example 2

[0026] Construction of implementation case 2 recombination cassette

[0027] (1) Put Buffer and restriction endonucleases on ice first, prepare a double enzyme digestion system, and place them at 30°C for 30 minutes. After the enzyme digestion is completed, the enzyme digestion product is purified and recovered.

[0028] (2) Insert the obtained target gene into the pYPGE15 plasmid that has been transformed into the KanMX gene with BamH I as the restriction site in turn, and construct the expression vectors pYPGE15_KanMX_Aro9 (S.c) and pYPGE15_KanMX_Aro9 (S.b). The amplification PCR diagram is shown in figure 2 . The expression vector was verified by single and double digestion to confirm that the expression vector was successfully constructed.

[0029] (3) Purify and recover the recombination cassette fragments of pYPGE15_KanMX_Aro9(S.c) and pYPGE15_KanMX_Aro9(S.b). For the construction of the recombination cassette, see image 3 , transformed into brewer's yeast by lithiu...

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Abstract

The invention aims to construct a beer yeast strain for overexpression of aromatic amino acid transaminase IIAro9. The method comprises the following steps: (1) thallus culture and beer yeast genome acquisition: firstly, dividing beer yeast TCCC 31194 into three regions for culture, and then treating the thallus with SDS to obtain a beer yeast genome; (2) acquisition of a target gene segment: designing a reasonable homologous primer, and amplifying Aro9(S.c) and Aro9(S.b) target genes through a PCR technology; (3) construction of a gene overexpression component: constructing pYPGE15_KanMX_Aro9(S.c) and pYPGE15_KanMX_Aro9(S.b) expression vectors by taking a pYPGE15 plasmid as a vector; (4) acquisition of a recombinant strain: transferring the pYPGE15_KanMX_Aro9 expression vectors into the beer yeast TCCC 31194 to obtain recombinant yeast of TCCC 31194::Aro9(S.c) and TCCC 31194::Aro9(S.b); (5) beer fermentation: culturing successfully-transformed yeast strains for 10 days under proper conditions; and (6) detecting the contents of triol tetraester, aromatic alcohol and aromatic amino acid and related fermentation performance.

Description

technical field [0001] The invention belongs to the field of genetic engineering and mainly introduces the construction of a recombinant system for overexpressing aromatic amino acid transaminase II Aro9 in brewer's yeast, thereby achieving the purpose of improving the contents of triol tetraesters, aromatic alcohols and aromatic amino acids in beer. Background technique [0002] As the main flavor substance in beer, higher alcohol is very important to the formation of the overall flavor of beer. Higher alcohol, also known as miscellaneous alcohol, refers to alcohols containing more than 3 carbon atoms. An appropriate amount of higher alcohol can make the taste of beer soft and harmonious, and the wine body is round and mellow. However, if the content of higher alcohol in beer is too high, it will have an unpleasant taste. bitterness. Usually isoamyl alcohol is the higher alcohol with the highest content in beer. Excessive isoamyl alcohol and isobutanol will make beer have ...

Claims

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Application Information

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IPC IPC(8): C12N15/04C12N9/10C12N15/81
CPCC12N9/1096C12N15/81C12Y206/01057
Inventor 钟成杨颖崔吉晓
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY