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Saccharomyces cerevisiae for industrial production with high organic acid tolerance and construction method thereof

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae strains, which is applied in the field of Saccharomyces cerevisiae strains for industrial production and its construction, can solve the problems of product quality reduction, harm to Saccharomyces cerevisiae physiology and metabolism, and affect the quality of fermentation products, and achieve the goal of improving tolerance Effect

Active Publication Date: 2022-06-21
SHAOXING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The severe low pH pressure caused by a large amount of organic acids produced during the fermentation process will adversely affect the activity of yeast, endanger the physiology and metabolism of Saccharomyces cerevisiae, prolong the fermentation time, and even affect the quality of the fermentation product, resulting in the final product serious loss of quality

Method used

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  • Saccharomyces cerevisiae for industrial production with high organic acid tolerance and construction method thereof
  • Saccharomyces cerevisiae for industrial production with high organic acid tolerance and construction method thereof
  • Saccharomyces cerevisiae for industrial production with high organic acid tolerance and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Construction of a PrA-encoding allele knockout and a PrA-encoding allele-modified strain

[0068] (1) Knockout of PrA encoding allele

[0069] ① Using the plasmid pUG6 as the template, PCR amplify the kanamycin-resistant fragment, add homology arms before and after the sequence, for the PrA coding allele PEP4 (the full-length sequence in the genome is SEQ ID NO: 1 , including the knockout of the original 1kb promoter coding region and the original 1kb terminator coding region), the amplified DNA fragment is "REC-Kan";

[0070] Upstream primer Rec-F:

[0071] 5'-GTATTTAATCCAAATAAAATTCAAACAAAAACCAAAACTAACATGCAGCTGAAGCTTCGTACGC-3', SEQ ID NO: 3;

[0072] Downstream primer Rec-R:

[0073] 5'-ATGGCAGAAAAGGATAGGGCGGAGAAGTAAGAAAAGTTTAGCTCGCATAGGCCACTAGTGGATCTG-3', SEQ ID NO: 4

[0074] Bold parts are homology arms

[0075] The PCR reaction conditions were: 94°C for 5 minutes, 94°C for 30 seconds, 58°C for 40 seconds, 72°C for 1 minute and 50 seconds (30 cycles), and 72°C ...

Embodiment 2

[0131] Comparison of organic acid tolerance in liquid culture

[0132] Saccharomyces cerevisiae strains SWY85, SWY85F and SWY-ZH were activated and cultured in YPD liquid medium with natural acidity, and the culture conditions were 30°C and 160rpm for 48 hours. YPD medium and synthetic nitrogen-limited medium (SD medium) were adjusted to pH 30 and 27 with citric acid, tartaric acid, and malic acid, respectively, and the strains that had been previously activated with natural acidity YPD liquid medium were inoculated at 01% inoculum size Cells were cultured in YPD liquid medium with natural acidity and in liquid YPD medium with pH adjusted to 30 and 27 using organic acid, and cultured at 30°C and 160rpm to the middle and late logarithmic growth period, and the cell growth amount (OD) was determined. 600 value), and the amount of cell growth (OD) in YPD medium with natural acidity for the same time period 600 value) is the control, and the calculation of the cell growth inhibit...

Embodiment 3

[0134] Comparison of organic acid tolerance in solid culture

[0135] Preparation of medium: Refer to the liquid culture acid tolerance part of the experiment to prepare the medium. The solid medium needs to add 2% agar to the liquid medium for solidification. After adjusting the pH of the solid YPD medium to 30 and 27 with citric acid, tartaric acid and malic acid, respectively, the plate was poured out for use.

[0136]Centrifuge at 5000rpm to collect the cell culture medium that was cultured in liquid YPD medium at 28°C-30°C and 160rpm for 48 hours, wash the bacterial cells twice with sterile water and suspend them to the same cell concentration, press After the multiples of 10 were diluted by gradient concentration, 3 microliters were inoculated on the pre-prepared organic acidified solid medium, and after static culture at 28°C-30°C for 48-72 hours, the colony formation was observed, and after constructing strains and wild comparison between strains, such as Figure 13 ...

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Abstract

The invention discloses a Saccharomyces cerevisiae strain for industrial production with high organic acid tolerance and a construction method thereof. The construction method comprises knocking out one protease A (PrA) coding allele, and coding the second PrA, etc. Sequence recombination transformation of bit genes; the recombinant strain constructed by the present invention has significantly improved the tolerance to organic acids, compared with the original strain, under the YPD culture condition of pH 3.0, the growth inhibition rate of citric acid on the recombinant strain has been reduced 33.0%, tartaric acid reduced the growth inhibition rate of recombinant bacterial strains by 26.0%, and malic acid reduced the growth inhibition rate of recombinant bacterial strains by 77.1%; meanwhile, the recombinant bacterial strains constructed by the present invention showed higher than original bacterial strains in anaerobic fermentation experiments. Ethanol fermentation production capacity; the present invention provides a new idea and method for solving the inhibition of organic acids on fermentation without adding acid reducing agents and without changing the production process, and has advantages in stabilizing industrial production, reducing production costs and environmental protection Practical value and guiding significance.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the genetic recombination of industrial microorganisms, in particular to a Saccharomyces cerevisiae strain for industrial production with high organic acid tolerance and a construction method thereof. [0002] technical background [0003] In the process of yeast fermentation, a large amount of organic acid will be produced, which will increase the acidity of the fermentation system and reduce the pH value. Due to the limitation of acid resistance of Saccharomyces cerevisiae, the increase of acidity in fermentation mash will reduce the fermentation performance of yeast and affect the production. For some fermented foods (wine, bread, soy sauce and vinegar, etc.), organic acids are also important flavor substances and flavor precursors, which are indispensable for the improvement of food flavor quality. In the process of yeast fermentation production, due to the continuous ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/57C12R1/865
CPCC12N9/60C12N15/81C12N2800/30
Inventor 张洪波从剑杭黄燕陈敏芳何汶霞李燕婷何欣煜陆佳燕
Owner SHAOXING UNIVERSITY
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