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Low-secretion protease a saccharomyces cerevisiae strain and construction method thereof

A technology of Saccharomyces cerevisiae strain and protease, which is applied in the field of bioengineering, can solve the problems of abnormal temperature sensitivity of bacteria, temperature sensitivity, and influence on the growth and polarity transport of reproductive cells, so as to increase the fermentation speed, improve the foam retention, fermentation performance and good growth performance

Active Publication Date: 2017-05-03
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that knocking out the SEC5 gene in Drosophila will affect the growth of germ cells and the polar transport of the membrane in the cell, and make it sensitive to temperature; knocking out the SEC5 gene in yeast will cause the bacteria to respond to temperature. unusually sensitive
[0008] The method of reducing the secretion of protease A under stress conditions by knocking out the key protein factor Sec5p in the constitutive secretion pathway of Saccharomyces cerevisiae has not been reported yet

Method used

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  • Low-secretion protease a saccharomyces cerevisiae strain and construction method thereof
  • Low-secretion protease a saccharomyces cerevisiae strain and construction method thereof
  • Low-secretion protease a saccharomyces cerevisiae strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Construction of low-secretion protease A Saccharomyces cerevisiae strain WS5

[0068] The starting strain W303-1A was constructed by two homologous recombination methods to construct recombinant genetically engineered strains.

[0069] According to the yeast genome data in Genebank and the integrated plasmid sequence, the primers in the following examples were designed.

[0070] Table 1 Primers used in this example

[0071]

[0072]

[0073] Note: The underlined part indicates the enzyme cutting site

[0074] (1) Construction of recombinant plasmid pUC-AKB

[0075] The construction process of the recombinant plasmid pUC-AKB is as follows: figure 1 shown.

[0076] ① Using the total DNA of the yeast strain W303-1A as a template, the upstream sequence SA of the SEC5 gene was amplified by PCR with primers SA1 and SA2;

[0077] PCR reaction conditions: 95°C for 5min; 94°C for 45s; 61°C for 45s; 72°C for 30s, 30 cycles; 72°C for 10min, 0.8% agarose gel e...

Embodiment 2

[0100] Example 2: Construction of low-secretion protease A Saccharomyces cerevisiae strain SS5-2

[0101] The starting strain S6 was constructed by two homologous recombination methods to construct recombinant genetically engineered strains. The primers used in this example are the same as those in Example 1.

[0102] (1) Knockout of one allele of SEC5

[0103] Using the recombinant plasmid pUC-AKB obtained in Example 1-(1) as a template, the recombinant gene cassette SA-KanMX-SB fragment was obtained by PCR amplification. Using the method described in Example 1-(2), the recombination cassette was introduced into the starting strain S6 to obtain an allelic knockout recombinant strain SS5K-1. Extract the genome of SS5K-1 as a template, and carry out PCR amplification with primers S1U / S1D and S2U / S2D respectively to obtain 1276bp (nucleotide sequence as shown in SEQ ID NO: 2) and 1715bp (nucleotide sequence The band shown in SEQ ID NO: 3); and the band of 1276bp and 1715bp ca...

Embodiment 3

[0110] Embodiment 3: Fermentation experiment of low-secretion protease A Saccharomyces cerevisiae WS5

[0111] The starting strain W303-1A and the recombinant Saccharomyces cerevisiae laboratory strain WS5 were simultaneously subjected to beer fermentation experiments. Nitrogen source deficient wort production process: Weigh a certain amount of crushed malt, saccharify at 65°C according to the material-water ratio of 1:4 until the iodine test is completed, adjust the apparent sugar content to 5°Brix with a sugar meter, and then add glucose to The appearance Brix is ​​10°Brix. Centrifuge the cultivated seed solution to obtain yeast slurry, put it into nitrogen-deficient wort medium according to the inoculum size of 0.8%, and let it stand for fermentation at 16° C. for 5 days.

[0112] (1) Protease A activity and bubble retention of low-secretion protease A strain WS5

[0113] The foam persistence of the starting strain W303-1A and the recombinant strain WS5 was determined by ...

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Abstract

The invention discloses a saccharomyces cerevisiae strain secreted by low protease A and a construction method thereof, and the saccharomyces cerevisiae strain can improve the foam stability of beer. The accharomyces cerevisiae strain is obtained by knocking out the complete sequence of SEC5 gene of a key protein factor Sec5p in a coding constitutive secretory pathway. The accharomyces cerevisiae strain is fermented for 4-5d at the temperature of 16 DEG C in wort lacking a nitrogen source, the enzyme activity of protease A and original strains in a fermentation broth are obviously reduced, and the foam retention is obviously increased; the fermenting performance and the growth performance are good, and the condition of affecting the growth performance of recombination strains or temperature lethal does not appear. For a type strain W303-1A, the unexpected effect is obtained. The growth fermentation of the recombination strains is not reduced, the fermentation speed is increased, and the fermentation time is shortened from 5 days to 4 days.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of bioengineering, in particular to a low-secretion protease A brewing yeast strain and a construction method thereof. [0003] Background technique: [0004] Pure draft beer refers to a healthy, fashionable, fresh, natural, hygienic, safe and nutritious draft beer produced by aseptic brewing, aseptic filtration and aseptic packaging. Since there is no pasteurization or instant sterilization in the production process, the destruction of flavor substances and nutrients caused by heating is avoided, and the fresh taste and ingredients of beer are maintained. Therefore, compared with ordinary beer, pure draft beer is purer and healthier. Refreshing, fresher and more nutritious. [0005] However, pure draft beer has obvious quality defects in terms of foam quality, that is, the foam stability gradually decreases with the prolongation of shelf life. Therefore, improving the foam stability of pure draft b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/18C12N15/81C12N15/65C12C11/02C12Q1/68C12Q1/04C12R1/865
CPCC07K14/395C12C11/02C12Q1/6895
Inventor 陈叶福肖冬光刘明明郭学武董健张翠英杜丽平马立娟
Owner TIANJIN UNIV OF SCI & TECH
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