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A Yeast Strain with Low Protease A Excretion Under Stress Conditions

A technology of Saccharomyces cerevisiae strain and protease, which is applied in the field of bioengineering to achieve the effects of improving foam stability and reducing enzyme activity

Active Publication Date: 2018-10-16
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 Yeast Strain with Low Protease A Excretion Under Stress Conditions
  • A Yeast Strain with Low Protease A Excretion Under Stress Conditions
  • A Yeast Strain with Low Protease A Excretion Under Stress Conditions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of deletion mutant strains

[0044](1) Construction of pUC-VABK plasmid

[0045] The construction process of the recombinant plasmid pUC-VABK is as follows: figure 1 shown.

[0046] ①Using the total DNA of the yeast strain W303-1A as a template, the upstream sequence VA of the VPS10 gene was amplified by PCR;

[0047] Upstream primer VA-U: CCG GAATTC GAGTTTATGAGAAGGTTGGAGG (SEQ ID NO: 3)

[0048] Downstream primer VA-D: CGG GGTACC AAAGACCCGAGTAGTTGGAG (SEQ ID NO: 4)

[0049] The underlined part is the enzyme cleavage site;

[0050] PCR reaction conditions: 95°C for 5min; 94°C for 45s; 63°C for 1min; 72°C for 60s, 30 cycles; 72°C for 10min, 0.8% agarose gel electrophoresis to identify the amplified product;

[0051] PCR reaction system (20μL)

[0052]

[0053] The PCR product was connected to the pUC19 plasmid vector containing Amp resistance to obtain the recombinant plasmid pUC-VA.

[0054] ②Using the total DNA of the yeast strain ...

Embodiment 2

[0075] Embodiment 2: Construction of overexpression strain

[0076] (1) Construction of Yep-PVK plasmid

[0077] The construction process of the recombinant plasmid Yep-PVK is as follows: Figure 5 shown.

[0078] ① Use the pPGK1 plasmid as a template to amplify the strong promoter PGK1p-PGK1t gene fragment by PCR;

[0079] Upstream primer PGK-U: CGC GGATCC TCTAACTGAT CTATCCAAAACTGA (SEQ ID NO: 13)

[0080] Downstream primer PGK-D: CGC GTC GAC TAACGAACGCAGAATTTTC (SEQ ID NO: 14)

[0081] The underlined part is the enzyme cutting site

[0082] PCR reaction conditions: 95°C for 5min; 94°C for 45s; 61°C for 1min; 72°C for 100s, 30 cycles; 72°C for 10min, 0.8% agarose gel electrophoresis to identify the amplified product;

[0083] The PCR product was connected to the expression vector YEP352 to obtain the recombinant plasmid Yep-P.

[0084] ② Using the total DNA of the yeast strain W303-1A as a template, the VPS10 gene was amplified by PCR;

[0085] Upstream primer VPS1...

Embodiment 3

[0102]Embodiment 3: beer fermentation experiment

[0103] Taking the recombinant strains obtained in Examples 1 and 2 and the starting strain W303-1A as experimental objects,

[0104] (1) Seed cultivation

[0105] 1) Activation of strains: The preserved strains were transferred to YEPD inclined test tubes at 30°C for two days of activation.

[0106] 2) Primary seed culture: Take a loop of slant bacteria, inoculate it into a test tube containing 5 mL of wort medium, and culture it at 30° C. and 180 rpm for 14 hours.

[0107] 3) Secondary seed cultivation: The primary seed liquid was transferred into a 150mL Erlenmeyer flask containing 50mL wort according to the inoculum amount of 10%, and cultured at 16° C. for 36 hours.

[0108] (2) Beer fermentation

[0109] Fermentation experiment: The secondary seed liquid was put into a 250mL Erlenmeyer flask filled with 150mL 10Brix nitrogen-deficient wort according to the inoculum amount of 10%, covered with a fermentation plug to kee...

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Abstract

The invention provides a new method for regulating the extracellular and extracellular secretion of protease A under stress conditions, which is realized by overexpressing the receptor gene VPS10 that controls the sorting of protease A from the Golgi body to the vacuole. In the strain overexpressing VPS10, the intracellular enzyme activity increased by 1.47 times, while the extracellular enzyme activity decreased to 61% of the original strain, and the fermentation characteristics of the overexpressed strain did not change significantly. The invention provides a new idea and method for reducing the extracellular enzyme activity of protease A without changing the fermentation characteristics, and has guiding significance for improving the foam stability of industrial yeast.

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): C12N1/16C12N15/81C12C11/00C12Q1/6895C12Q1/04C12R1/865
CPCC12C11/00C12N15/81C12Q1/6895C07K14/39
Inventor 陈叶福肖冬光韩月然郭学武董健张翠英杜丽平马立娟
Owner TIANJIN UNIV OF SCI & TECH
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