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Method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1

A Saccharomyces cerevisiae and gene technology, applied in the field of bioengineering, can solve problems such as protein oxidative damage, yeast toxicity, and increased ROS level, and achieve the effect of improving viability and improving the ability

Active Publication Date: 2019-12-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the fermentation process, the concentration of pyruvate is too high, which is toxic to the yeast, and the high acid concentration will lead to an increase in the level of intracellular ROS, causing oxidative damage to DNA and proteins.

Method used

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  • Method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1
  • Method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1
  • Method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of deletion mutant strains

[0037] Using the wild-type Saccharomyces cerevisiae genome as a template, and using P1 / P2, P3 / P4, and P5 / P6 as primers, respectively, the left arm (L), histidine gene (M) and right arm of the gene to be knocked out were amplified. (R), the knockout frame REV1-LMR was constructed by fusion PCR ( figure 1 ). The correctly sequenced knockout frame was introduced into the starting strain Saccharomyces cerevisiae BY4741 by chemical transformation, the positive transformants were screened using the histidine tag gene, and the genome was extracted for PCR sequencing verification.

[0038] Table 1 Primer sequence list

[0039]

Embodiment 2

[0040] Embodiment 2: Construction of overexpression strain

[0041] Using the genome of Saccharomyces cerevisiae BY4741 as a template and P7 / P8 as primers to amplify the target gene REV1, digest the amplified product with the same restriction enzymes EcoRI and XhoI as the plasmid PY26, and connect the gene REV1 to PY26 by T4 ligase , Transcription and translation are initiated by the strong promoter GPD1, positive transformants are screened using the URA3 gene on the recombinant plasmid, and finally the plasmid is extracted for verification to obtain the overexpression strain Rev1Δ / REV1.

Embodiment 3

[0042] Embodiment 3: the mensuration of each bacterial strain growth performance

[0043] (1) Plate growth experiment: inoculate a single colony of the strain to be tested in 20 mL of YNB (0.67% YeastNitrogen Base without Amino Acids, 2% Glucose) liquid medium for overnight activation, then transfer to YNB medium and cultivate until After several phases, measure the bacterial concentration and adjust the bacterial suspension to OD 600 = 1.0, using this as the initial concentration, carry out 5 times of 10-fold gradient dilution, and sequentially plant 3 μ L of bacterial liquid on the corresponding solid YNB medium, cultivate at 30 ° C for 2-3 days, observe the growth of the bacteria and take pictures ( figure 2 ).

[0044] (2) Growth curve test: inoculate a single colony of the strain to be tested in 20 mL of YNB (0.67% YeastNitrogen Base without Amino Acids, 2% Glucose) liquid medium for overnight activation, and then transfer to the corresponding YNB liquid medium In, con...

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Abstract

The invention discloses a method for regulating saccharomyces cerevisiae oxygen stress through Y-family polymerase Rev1, and belongs to the technical field of bioengineering. A gene REV1 is knocked out through a homologous recombination method, a knock-out strain rev1 delta is constructed, thus the oxidation stress resisting ability of a yeast strain is lowered, specifically, under the stress condition, the biomass of the knout-out strain is decreased by 9%, the survival rate is decreased by 34.8%, the gene mutation frequency is decreased by 63.8%, and the yield of pyruvic acid is decreased by30.8%. The gene REV1 is over-expressed through pY26 plasmid, an overexpression strain rev1 delta / REV1 is constructed, thus the oxidation stress resisting ability of the strain is enhanced, specifically, the biomass is increased by 8.3%, the survival rate is increased by 11.5%, the gene mutation frequency is increased by 186.2%, and the yield of the pyruvic acid is increased by 57.7%. The resultsshow that Rev1 can improve the survival ability and fermentation performance of the yeast strain under the oxygen stress condition.

Description

technical field [0001] The invention relates to a method for regulating the oxygen stress of Saccharomyces cerevisiae by using Y family polymerase Rev1, belonging to the technical field of bioengineering. Background technique [0002] During the process of growth and metabolism, microorganisms are easily attacked by various exogenous and endogenous environments. The exogenous environment mainly includes ionizing radiation, ultraviolet radiation and various chemical reagents. grade metabolites. Most of these factors will cause the accumulation of reactive oxygen species in cells, causing oxidative damage to intracellular DNA and hindering cell growth and metabolism. In industrial applications, Saccharomyces cerevisiae can be used not only to produce alcohol, but also to produce organic acids, proteins, etc. In the field of life sciences, Saccharomyces cerevisiae is highly homologous to humans, and the analysis of Saccharomyces cerevisiae genes can improve the diagnosis and ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/40C12R1/865
CPCC12N9/1241C12N15/81C12P7/40
Inventor 吴静姚瑞刘立明陈修来刘佳罗秋玲
Owner JIANGNAN UNIV
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