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Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain

A technology of Saccharomyces cerevisiae strain and protease, which is applied in the field of bioengineering to achieve the effect of improving foam retention

Active Publication Date: 2015-03-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under stress conditions such as lack of various nutrients and insufficient nitrogen sources in the later stage of fermentation, it will be transported to the outside of the cell by mistake and activated outside the cell.

Method used

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  • Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of PEP4 gene knockout and MRL1 gene overexpression strain

[0045] (1) Construction of Yep-PMKZ plasmid

[0046] The construction process of recombinant plasmid Yep-PMKZ is as follows figure 1 Shown.

[0047] ① Using pPGK1 plasmid as a template, PCR amplified a strong promoter PGK1p-PGK1t gene fragment;

[0048] Upstream primer PGK-U: CGC GGATCC TCTAACTGAT CTATCCAAAACTGA (SEQ ID NO: 3)

[0049] Downstream primer PGK-D: CGC GTCGAC TAACGAACGCAGAATTTTC (SEQ ID NO: 4)

[0050] The underlined part is the restriction site

[0051] PCR reaction conditions: 95℃5min; 94℃45s; 61℃1min; 72℃100s, 30 cycles; 72℃10min, 0.8% agarose gel electrophoresis to identify amplified products;

[0052] PCR reaction system (20μL)

[0053] PCR buffer

dNTP

Upstream and downstream primers

Template

Taq enzyme

DdH2O

total capacity

2.0μL

1.5μL

1.0μL each

1.0μL

0.5μL

13.0μL

20.0μL

[0054] The PCR product was ligated to the expression vector YEP352 to obtain the recombina...

Embodiment 2

[0088] Example 2: Comparison of intracellular and extracellular protease A activity

[0089] Two strains of yeast strains RY1 and RPMKZ were used for beer fermentation experiments. After the fermentation, the fermentation broth was centrifuged to collect the bacteria. After the cell wall was broken, the intracellular protease A activity was measured, and the extracellular protease A activity of the fermentation broth was measured at the same time. The results are shown in Table 1. Through observation, it was found that the intracellular enzyme activity of the recombinant strain RPMKZ that overexpressed the MRL1 gene while a single PEP4 gene was deleted was not much different from that of the original strain, but the extracellular enzyme activity was reduced to 54% of the original strain. This is because although knocking out a copy of the PEP4 gene reduces the total expression of protease A, overexpression of the vacuolar sorting receptor gene MRL1 on the basis of knocking out PE...

Embodiment 3

[0092] Example 3: Fermentation performance-comparison of α-amino nitrogen changes

[0093] α-amino nitrogen is the main nitrogen source for yeast growth and metabolism. This study investigated the difference in the assimilation and metabolism of α-amino nitrogen between yeast strains RY1 and RPMKZ. Such as Figure 5 As shown, from the beginning of the main fermentation, the concentration of α-amino nitrogen in the fermentation broth showed an obvious decreasing trend, and at the end of fermentation, the change trend of α-amino nitrogen tended to be stable. This is because in the main fermentation period, yeast growth and metabolism use a large amount of α-amino nitrogen, so the rate of assimilation and absorption is very fast. At the end of fermentation, the number of yeast reached a stable value, and the assimilation rate of α-amino nitrogen also stabilized. Studies have shown that there is not much difference in the concentration of α-amino nitrogen between the two strains dur...

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Abstract

The invention provides a new method for regulating intra-cellular and extra-cellular secretion of proteases A under the stress condition. The regulation is realized through over-expressing of an MRL1 gene for coding a vacuolar sorting receptor during knocking-out of an allele of a protease A coding gene, that is, a PEP4 gene. With the adoption of the bred saccharomyces cerevisiae strain, the extra-cellular secretion amount of the proteases A is obviously decreased, the foam retention of draft beer can be improved, the extra-cellular enzyme activity of the proteases A is obviously lower than that of an original strain and is decreased to about 54% of that of the original strain, and meanwhile, compared with the intra-cellular enzyme activity of the original strain, the intra-cellular enzyme activity of the proteases A of the recombinant strain is not changed significantly. A new idea and a new method are provided for reduction of the extra-cellular enzyme activity of the proteases A without change of fermentation characteristics and have the guiding significance in improving the industrial yeast foam stability.

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 yeast strain suitable for low protease A exocrine under stress conditions at the end of fermentation and a construction method thereof. Background technique: [0002] During the development of pure draft beer, some quality problems related to pure draft beer have been plagued by brewing engineers, among which the foam stability problem of pure draft beer is particularly prominent. Since pure draft beer is not pasteurized before packaging, at the end of fermentation, the nitrogen source is insufficient, and the high alcohol and carbon dioxide concentration will create a stress environment for the yeast, leading to the secretion of protease A, decomposing the foam-positive protein, and reducing the beer. The foam retention has an adverse effect on the foam quality of pure draft beer. Therefore, improving the foam stabi...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12C11/00C12R1/865
CPCC07K14/395C12C11/00C12C2200/05C12N9/60
Inventor 陈叶福肖冬光韩月然龚瑞郭学武董健张翠英杜丽平马立娟
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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