Kluyveromyces marxianus-derived alkyl peroxide reductase and thioredoxin reductase and application thereof

A technology of alkyl peroxide and thioredoxin, applied in the application of improving the stress tolerance of Saccharomyces cerevisiae, in the field of gene and amino acid sequence of thioredoxin reductase KmTrxR, can solve limited problems and less functional research And other issues

Active Publication Date: 2016-05-25
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are very limited reports on Kluyveromyces m...

Method used

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  • Kluyveromyces marxianus-derived alkyl peroxide reductase and thioredoxin reductase and application thereof
  • Kluyveromyces marxianus-derived alkyl peroxide reductase and thioredoxin reductase and application thereof
  • Kluyveromyces marxianus-derived alkyl peroxide reductase and thioredoxin reductase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction of Saccharomyces cerevisiae strains overexpressing alkyl peroxide reductase KmTPX1

[0077] 1. Cloning of KmTPX1 gene

[0078] PrimerPremier5.0 primer design software was used to design the upstream primer TPX1-F, 5'-CTTGAGCTCAATGTCTCGTCTCGTCTCGT-3' and downstream primer TPX1 based on the complete sequence of the alkyl peroxide reductase KmTPX1 gene of K.marxianus (obtained by transcriptome sequencing) -R, 5'-TCCCCGCGGGGCTAAGCCAATAACTTATT-3', introducing a SacI restriction site at the 5' end and a SacII restriction site at the 3' end, respectively. The K. cicerisporus CBS4857 genome was used as a template for PCR amplification reaction. Among them, the PCR reaction system (50μL):

[0079]

[0080] PCR reaction conditions:

[0081]

[0082] After the PCR reaction, 2 μL of the PCR product was taken for agarose gel electrophoresis detection, and the product contained a total of 1093 nucleotides, including a 478 bp promoter and a 594 bp codin...

Embodiment 2

[0115] Example 2 Construction of a Saccharomyces cerevisiae strain overexpressing thioredoxin reductase KmTrxR

[0116] Similarly, using PrimerPremier5.0 primer design software, based on the complete sequence of K.marxianus thioredoxin reductase KmTrxR (acquired by transcriptome sequencing), the upstream primer TrxR-F, 5'-TCCGAGCTCCCCATGTCAAATGATGAAACG-3' and downstream primer TrxR were designed -R, 5'-TCCCCGCGGACGAGGAACCAACCTTTATT-3', introducing a SacI restriction site at the 5' end and a SacII restriction site at the 3' end, respectively. The K. cicerisporus CBS4857 genome was used as a template for PCR amplification reaction. The PCR reaction system and reaction conditions were the same as in Example 1, and the obtained PCR product contained a total of 1472 nucleotides, including its own promoter of 497 bp and a coding sequence of 960 bp.

[0117] The purified PCR product was combined with Saccharomyces cerevisiae multi-copy vector pRS425 ( figure 2 ) for ligation react...

Embodiment 3

[0119] Embodiment 3 Real-time quantitative PCR verifies the expression intensity of KmTPX1 gene and KmTrxR gene

[0120] In order to confirm that the recombinant plasmids containing KmTPX1 gene and KmTrxR gene constructed in Examples 1 and 2 can be successfully transcribed in Saccharomyces cerevisiae, we used real-time quantitative PCR to verify the expression intensity of these two genes.

[0121] First, the expression of the KmTPX1 gene was verified. The overexpressed KmTPX1 Saccharomyces cerevisiae strain (named TPX1) and the control yeast strain (named 423) containing the empty plasmid pRS423 were both cultured in SD medium lacking histidine (0.67% yeast nitrogen base, 2% glucose and other essential amino acid nutrients, SD-His). The two activated yeasts were respectively inoculated into SD-His medium according to the inoculum amount of 1% (v / v), and cultured at 30° C. and 150 rpm until the logarithmic growth phase (16-18 h). Cells were collected for total RNA extraction...

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Abstract

The invention belongs to the technical field of biomedicine and relates to kluyveromyces marxianus-derived alkyl peroxide reductase and thioredoxin reductase and an application thereof in improving stress resistance. The KmTPX1 gene of alkyl peroxide reductase gene contains 594 nucleotides, and 197 amino acids are coded; and the KmTrxR gene contains 960 nucleotides, and 319 amino acids are coded. Meanwhile, the KmTPX1 gene and KmTrxR gene can improve the tolerance of yeast cells against multiple inhibitors or stress factors, such as one or more of hydrogen peroxide, formic acid, acetic acid, furfural, ethanol, sodium chloride, phenol and guaiacol. Due to the improvement of the tolerance against formic acid, acetic acid, furfural, ethanol and salt ion concentration, the alkyl peroxide reductase gene and thioredoxin reductase gene play a crucial role in constructing a good cellulosic ethanol fermentation strain.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering applications, and specifically relates to the gene and amino acid sequence of alkyl peroxide reductase KmTPX1 derived from Kluyveromyces marx and the gene and amino acid sequence of thioredoxin reductase KmTrxR. At the same time, the present invention also relates to the application of the above two enzymes in improving the stress tolerance of Saccharomyces cerevisiae. Background technique [0002] Yeast has long been a microorganism that has been widely concerned by people, and it can be widely used in various industrial fields such as food, medicine, and brewing. Saccharomyces cerevisiae, in particular, has always been one of the most widely studied yeasts because of its important initial application in the brewing industry, whether it is the traditional brewing industry, the emerging bioethanol, or even the field of genetic engineering. The applied research of Saccharomyces cerevis...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N9/02C12N15/53C12N15/81C12N1/19C12R1/865
CPCC12N9/0051C12N9/0065C12Y108/01009C12Y111/01015
Inventor 袁文杰高教琪白凤武
Owner DALIAN UNIV OF TECH
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