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Method for reducing yellow wine yeast urea accumulation by regulating and controlling activating transcription factors

A technology of transcription activator and rice wine yeast, which is applied in the field of genetic engineering, can solve problems affecting the safety of rice wine products and achieve the effect of important industrial application prospects

Active Publication Date: 2019-03-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The accumulated urea will spontaneously react with ethanol in the system to generate 2A potential carcinogen ethyl carbamate, which seriously affects the safety of rice wine products

Method used

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  • Method for reducing yellow wine yeast urea accumulation by regulating and controlling activating transcription factors
  • Method for reducing yellow wine yeast urea accumulation by regulating and controlling activating transcription factors
  • Method for reducing yellow wine yeast urea accumulation by regulating and controlling activating transcription factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 The construction of rice wine yeast suitable for rapamycin-mediated regulatory protein subcellular localization

[0030] (1) Split the diploid Saccharomyces cerevisiae strain XZ-11 used for rice wine production to obtain haploid strains

[0031] According to the paper "Wu D H, Li X M, Shen C, et al.Isolation of a haploid from anindustrial Chinese rice wine yeast for metabolic engineering manipulation[J].Journal of the Institute of Brewing,2013,119(4):288-293 The method and steps described in the method and steps to obtain the haploid XZ-11a strain without resistance gene.

[0032] (2) Construction of auxotrophic haploid rice wine yeast JNZ01

[0033] Using the genome of rice wine yeast XZ-11 strain as a template, the upstream and downstream 300 bp sequences of the URA3 gene were respectively amplified, and the above two amplified fragments were fused by fusion PCR to obtain the URA3 gene knockout frame. The URA3 knockout frame was transformed into the XZ-11a...

Embodiment 2

[0042] Example 2 Fusion PCR construction fusion expression GLN3, FKBP12 and GAT1, FKBP12 recombination cassette

[0043] (GGGGS) was first transformed by whole-plasmid PCR 3 After the linker was introduced into the BamH I site on the high-copy plasmid vector pRS426-TEF-URA3, pRS426-TEF-GS-URA3 was obtained. The FKBP12 protein whose N-terminus was fused with four SV40 nuclear localization sequences was obtained by gene synthesis, and cloned into the EcoR I and Xho I sites of the vector pRS426-TEF-GS-URA3 by restriction enzyme digestion to obtain pRS426-TEF-GS-SV40-FKBP12 -URA3. The 500 bp sequences without stop codon at the end of GLN3 and GAT1 were respectively amplified from the genome, and cloned into the Spe I and BamH I sites of the vector pRS426-TEF-GS-SV40-FKBP12-URA3 by restriction enzyme digestion, respectively, to obtain pRS426 - TEF-Gln3D-GS-SV40-FKBP12-URA3 and pRS426-TEF-Gat1D-GS-SV40-FKBP12-URA3. Then, PCR amplification was performed using the obtained plasmid ...

Embodiment 3

[0046] Example 3 eliminates the CRISPR-Cas9 system plasmid

[0047] The positive transformants obtained by screening were subcultured in YPD medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), and were transferred to fresh YPD medium with 10% inoculum after every 24 hours of cultivation. . At the same time of each transfer, the 5-FOA and 5-FAA plates (YNB 1.7g / L, ammonium sulfate 5g / L, glucose 20g / L, uracil 25mg / L, tryptophan 25mg / L, 5-FOA 1g / L, 5-FAA 1g / L, agar powder 20g / L) was streaked until transformants were obtained on 5-FOA and 5-FAA plates. It was verified by colony PCR that p426-Gln3sgRNA, p426-Gat1sgRNA and p414-TEF1p-Cas9-CYC1t had been eliminated in the transformants, and recombinant rice wine yeast JNZ02 (MATa, Δura3, Δtrp1, TOR1S1972R, Δfpr1, GLN3::FKBP12) and JNZ03 (MATa , Δura3, Δtrp1, TOR1S1972R, Δfpr1, GAT1::FKBP12).

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Abstract

The invention discloses a method for reducing yellow wine yeast urea accumulation by regulating and controlling activating transcription factors, and belongs to the field of genetic engineering. Recombinant yellow wine yeast JNZ01 (MATa, delta ura3, delta trp1,TOR1S1972R, delta fpr1) are used as original strains; GLN3, FKBP12, GAT1 or FKBP12 is subjected to fusion expression on a genome; SPT15 andFRB are subjected to fusion expression on plasmid; by adding rapamycin, FKBP12 and FRB are combined. On the basis, Gln3 and Gat1 are transferred to the cell nucleus under the effect of cell nucleus location protein Spt15; activation urea uses related gene transcription, so that the urea accumulation in the fermentation process is respectively reduced by 49.45 percent and 38.98 percent and is respectively reduced to 8.25 mg / L and 9.95mg / L.

Description

technical field [0001] The invention relates to a method for regulating transcription activating factors to reduce urea accumulation in rice wine yeast, which belongs to the field of genetic engineering. Background technique [0002] Saccharomyces cerevisiae has a preference for the utilization of nitrogen sources, preferentially utilizes the preferred nitrogen sources (such as glutamine, glutamic acid, etc.), and lags behind the utilization of non-preferred nitrogen sources (such as urea, proline, etc.). The phenomenon is known as the nitrogen metabolite repression effect. Regulators Gln3 and Gat1 are one of the global regulators of nitrogen metabolism repression in Saccharomyces cerevisiae, and they have transcriptional activation effects on genes related to nitrogen utilization. In the presence of preferential nitrogen sources, Gln3 and Gat1 were localized in the cytoplasm and could not enter the nucleus to activate the expression of genes related to non-preferential nit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/90C12G3/02C12R1/865
CPCC07K14/395C12G3/02C12N1/18C12N15/905
Inventor 周景文陈坚张伟平曾伟主夏小乐堵国成方芳
Owner JIANGNAN UNIV
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