Application of GSH synthesis and circulation related protein as well as recombinant saccharomyces cerevisiae strain

A technology for recombining Saccharomyces cerevisiae and Saccharomyces cerevisiae strains, applied in the field of microorganisms, can solve problems such as deletion

Pending Publication Date: 2021-09-14
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that D-EAA plays a certain role in stress resistance. Yeast strains lacking the gene ALO1, which encodes the enzyme that catalyzes the last step in the synthesis of D-EAA, have no effect on H 2 o 2 and O 2 -sensitive

Method used

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  • Application of GSH synthesis and circulation related protein as well as recombinant saccharomyces cerevisiae strain
  • Application of GSH synthesis and circulation related protein as well as recombinant saccharomyces cerevisiae strain
  • Application of GSH synthesis and circulation related protein as well as recombinant saccharomyces cerevisiae strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the acquisition of exogenous gene

[0048] The present invention provides 12 genes encoding GSH synthesis and cycle-related enzymes, which are respectively: the superoxide dismutase (SOD) gene sod1 derived from Kluyveromyces marxianus, and the γ-glutamylcysteine ​​synthetase (GSH1) encoding Gene gsh1, encoding glutathione synthase (GSH2) gene gsh2, encoding glutathione peroxidase (GPX) gene gpx.km, encoding cytoplasmic glutaredoxin (GRX) gene grx2, encoding NADH kinase (NADHK ) gene pos5, encoding glutathione oxidoreductase (GLR) gene glr1 in cytoplasm and mitochondria; gene gpx.wm encoding GPX from Wallemiamellicol, gene grx35095 encoding GRX, gene NK60672 encoding NADHK, encoding glutathione synthesis The gene gs61512 of the enzyme; the gene ttc0189 encoding SOD derived from Thermus thermophilus HB27. After codon optimization, twelve exogenous genes related to GSH synthesis and cycle were obtained by PCR.

Embodiment 2

[0049] Embodiment 2: Construction of recombinant Saccharomyces cerevisiae

[0050] Firstly, the fragment XhoI-TDH3p-HindIII-BamHI-GPM1t-SpeI was obtained by overlapping extension PCR technology, and then the fragment was digested with restriction endonucleases XhoI and SpeI, and the fragment was cloned by T4 connection and linearized with the same endonuclease On the vector pRS416, the plasmid cassette EGSH-HE was obtained. Afterwards, the obtained twelve exogenous genes were assembled by Gibson, cloned on the vector EGSH-HE linearized by HindIII and BamHI, and transformed into Escherichia coli competent Trans-T1. The positive clones were verified by E. coli colony PCR, and E. coli strains EGSH-1-12 were obtained. Extraction of large intestine plasmids 416-sod1, 416-ttc0189, 416-gsh1, 416-gsh2, 416-gs61512, 416-gpx.km, 416-gpx.wm, 416-grx2, 416-grx35095, 416-glr1, 416-pos5 and 416-nk60672 were transformed into Saccharomyces cerevisiae BY4741 respectively. Yeast strains YGSH...

Embodiment 3

[0051] Example 3: Strains resistant to H 2 o 2 representation of

[0052] Experimental Materials:

[0053] Strains YGSH-1-12 and BY4741;

[0054] experimental method:

[0055] SC medium: synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder lacking uracil, histidine, leucine and tryptophan 2g / L, histidine 0.1g / L, Leucine 0.1g / L, tryptophan 0.02g / L, 1M NaOH to adjust the pH to 6.0. (or adjust the pH to 3.0 with HCL);

[0056] YPD medium: glucose 20g / L, peptone 20g / L, yeast powder 10g / L.

[0057] Pick single colonies of YGSH-1~YGSH-12 and BY4741 from the solid streak plate and inoculate them in 3ml of corresponding SC auxotrophic liquid medium, and culture overnight at 30°C and 220rpm. Then with the initial OD 600 =0.2 Inoculate in fresh 5ml corresponding SC liquid medium, culture at 30°C and 220rpm for about 10-12h until the bacteria grow to the mid-logarithmic phase to obtain secondary seeds, then use the initial OD 600 = 0.2 Inoculated in...

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Abstract

The invention relates to the technical field of microorganisms, and discloses application of GSH synthesis and circulation related genes as well as a recombinant saccharomyces cerevisiae strain. The recombinant saccharomyces cerevisiae strain with excellent stress resistance is constructed by selecting GSH synthesis and circulation related genes from kluyveromyces marxianus, marine acid-resistant bacteria walleria mellica and extreme thermus thermophilus HB27. Compared with wild saccharomyces cerevisiae, the recombinant saccharomyces cerevisiae shows different effects in H2O2 resistance, heat shock resistance, high temperature resistance, acid resistance and salt resistance according to different exogenous genes; and an excellent yeast strain is provided for industrial fermentation production, and also a cell model is provided for later research on generation or protection of ROS in eukaryotes.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to the application of GSH synthesis and cycle-related proteins and recombinant brewer's yeast strains. Background technique [0002] Saccharomyces cerevisiae will produce excessive reactive oxygen species (ROS) under unfavorable living conditions such as high temperature, high salt, acidity and alkalinity, which will cause it to suffer oxidative damage and even lead to death. When Saccharomyces cerevisiae is exposed to ROS, it triggers intracellular oxidative stress, so its stress resistance is closely related to oxidative stress. The main site of ROS generation is mitochondria, which is the organelle with the most serious oxidative damage, so protecting mitochondria from oxidative damage is the focus of antioxidant defense. The antioxidant defense system of Saccharomyces cerevisiae consists of enzymatic defense system and non-enzymatic defense system. Antioxidant protective enzy...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N9/02C12N9/00C12N9/08C12N9/12C12N15/81C07K14/39C07K14/195C12R1/865
CPCC12N9/0089C12N9/93C12N9/0065C12N9/0051C12N9/1205C07K14/39C07K14/195C12N15/81C12Y115/01001C12Y603/02002C12Y603/02003C12Y111/01009C12Y108/01007C12Y207/01086
Inventor 元英进周蒙雨丁明珠王敏蓁
Owner TIANJIN UNIV
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