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A kind of exocrine yeast strain with low protease A under stress conditions and its construction method

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: 2017-08-29
TIANJIN UNIV OF SCI & TECH
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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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  • A kind of exocrine yeast strain with low protease A under stress conditions and its construction method
  • A kind of exocrine yeast strain with low protease A under stress conditions and its construction method
  • A kind of exocrine yeast strain with low protease A under stress conditions and its construction method

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 the recombinant plasmid Yep-PMKZ is as follows figure 1 shown.

[0047] ① Using the pPGK1 plasmid as the template, PCR amplified the 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 GTC GAC TAACGAACGCAGAATTTTC (SEQ ID NO: 4)

[0050] The underlined part is the restriction site

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

[0052] PCR reaction system (20μL)

[0053] PCR buffer

dNTP

Upstream and downstream primers

template

Taq enzyme

DdH2O

total capacity

2.0 μL

1.5μL

1.0 μL each

1.0μL

0.5μL ...

Embodiment 2

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

[0089] Beer fermentation experiments were carried out on two yeast strains RY1 and RPMKZ. After the fermentation, the fermentation broth was centrifuged to collect bacterial cells, and the intracellular protease A activity was measured after cell wall breaking treatment, and the extracellular protease A activity of the fermentation broth was measured at the same time. The results are shown in Table 1. It was found by observation that the intracellular enzyme activity of the recombinant strain RPMKZ overexpressing the MRL1 gene with a single PEP4 gene deletion was similar to that of the starting strain, but the extracellular enzyme activity was reduced to 54% of the starting strain. This is because although knocking out one copy of the PEP4 gene reduces the total expression of proteinase A, overexpression of the vacuolar sorting receptor gene MRL1 on the basis of knocking out PEP4 can increase...

Embodiment 3

[0092] Example 3: Fermentation Performance - Comparison of Alpha-Amino Nitrogen Changes

[0093] α-Amino nitrogen is the main nitrogen source for yeast growth and metabolism. This study investigated the differences in the assimilation and metabolism of α-amino nitrogen between yeast strains RY1 and RPMKZ. like Figure 5 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 the fermentation, the changing trend of α-amino nitrogen became 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 became stable. The study showed that the concentration of α-amino nitrogen of the two strains did not change much during the beer ferme...

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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 secretion under stress conditions at the end of fermentation and a construction method thereof. Background technique: [0002] During the development of pure draft beer, brewing engineers have been troubled by some quality problems related to pure draft beer, among which the foam stability 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 stressful environment for the yeast, resulting in the secretion of protease A, decomposing the foam-positive protein, and reducing the beer Foam retention, which adversely affects the foam quality of pure draft beer. Therefore, improving the foam stabil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 UNIV OF SCI & TECH
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