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Recombinant yeast cell and its preparation method and use

A yeast and cell technology, applied in the field of generating a recombinant yeast cell, can solve the problem of inability to completely ferment sugars and the like

Active Publication Date: 2020-04-07
FAR EASTERN NEW CENTURY COPRRATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Δgpd1Δgpd2 double-deleted Saccharomyces cerevisiae could not completely ferment sugars under anaerobic conditions

Method used

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  • Recombinant yeast cell and its preparation method and use
  • Recombinant yeast cell and its preparation method and use
  • Recombinant yeast cell and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Construction of recombinant vectors yTA-FPS-loxpKanMX, yTA-GPD1-loxpKanMX and yTA-GPD2-loxpKanMX

[0089]In order to use gene knockout technology to knock out (knock-out) target genes (that is to say fps1 gene, gpd1 gene and gpd2 gene) in the gene body of Saccharomyces cerevisiae BCRC 920077 (corresponding to DSM25508) Recombinant vectors yTA-FPS-loxpKanMX, yTA-GPD1-loxpKanMX and yTA-GPD2-loxpKanMX were respectively constructed in , and the construction process of these recombinant vectors is described in detail as follows.

[0090] A. Clone the upstream and downstream fragments of the target gene:

[0091] First, in order to clone the upstream fragment (hereinafter referred to as the Fps1-F fragment) and the downstream fragment (hereinafter referred to as the Fps1-R fragment) of the fps1 gene of S. nucleotide residue positions 49513 to 49703 and 52031 to 52180), the upstream fragment of the gpd1 gene (hereinafter referred to as the Gpd1-F fragment) and the ...

Embodiment 2

[0111] Embodiment 2. Construction of the recombinant vector puc-d-loxpKanMX-ENO1-psXDH with the xdh gene of Pichia stipitis

[0112] This example constructs a recombination with Delta sequence (Delta sequence), Loxp-KanMX-Loxp fragment, ENO1 promoter (ENO1 promoter), xdh gene of Pichia stipitis (hereinafter referred to as psXDH gene) and ENO1 terminator (ENO1 terminator) The vector puc-d-loxpKanMX-ENO1-psXDH was used to transform Saccharomyces cerevisiae BCRC 920077 and overexpress the psXDH gene, and its construction process is described in detail as follows.

[0113] A. Clone psXDH gene, Delta sequence, ENO1 promoter and ENO1 terminator respectively:

[0114] First, to clone the psXDH gene (corresponding to nucleotide residue positions 51 to 1142 shown in NCBI accession number XM_001386945.1) in Pichia stipitis BCRC 21775 (corresponding to ATCC 58376), and Saccharomyces Delta sequence in the retrotransposons of BCRC 920077 (corresponding to nucleotide residue positions 9694...

Embodiment 3

[0121] Embodiment 3. Construction of the recombinant vector pFENC-Cre with Cre recombinase gene

[0122]In this example, the construction of a recombinant vector pFENC-Cre with a Cre recombinase gene is generally based on the method described in Ulrich Güldener et al. (1996), Nucleic Acids Research, 24:2519-2524 conduct.

[0123] A. Gene synthesis of optimized Cre recombinase gene:

[0124] In order to obtain an optimized Cre recombinase gene that can be expressed in Saccharomyces cerevisiae, the applicant used the Cre recombinase gene (NCBI accession number: YP_006472.1) in Enterobacteria phage P1 (Enterobacteria phage P1) as the base Optimal adjustment, thereby obtaining the nucleotide sequence (1058bp) of the optimized Cre recombinase gene as shown in SEQ ID NO: 25. Then, 46 kinds of primers for synthesizing the optimized Cre recombinase gene were obtained through the analysis of the DNAWorks website (they are respectively shown in sequence identification numbers: 26 to 7...

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Abstract

The invention relates to a recombinant yeast cell as well as a preparation method and an application thereof. The invention discloses a method used for generating a recombinant yeast cell. The method comprises the following steps: providing parent yeast cells which can produce ethanol by consuming hexose and pentose, carrying out gene modification treatment on the parent yeast cells, wherein the gene modification treatment comprises deletion or destroy of an fps1 gene or disabling of the fps1 gene and introduction of a gene encoding xylitol dehydrogenase into genome DNA of the parent yeast cells, so that xylitol dehydrogenase can be excessively generated, and sequentially deleting or destroying a gpd1 gene and a gpd2 gene or sequentially disabling the gpd1 gene and the gpd2 gene. Therefore, the obtained recombinant yeast cell has excellent ethanol conversion ratio and relatively low byproduct generation rate.

Description

technical field [0001] The present invention relates to a method for producing a recombinant yeast cell, in particular to a method for genetically modifying a parental yeast cell that can produce ethanol by consuming six-carbon sugars and five-carbon sugars, so as to enhance its ethanol conversion rate and methods to reduce the rate of by-product formation. Background technique [0002] Generally, in the process of microbial fermentation, xylose is mainly converted into ethanol through the following two ways: [0003] (1) Oxidation-reductase pathway (oxido-reductase pathway), which includes the following steps: use xylose reductase (xylose reductase, XR) to reduce xylose to xylitol (xylitol), and then remove xylitol with xylitol Xylitol dehydrogenase (XDH) is used to phosphorylate xylitol into xylulose (xylulose), and then xylulose kinase (XK) is used to convert xylulose into xylulose-5-phosphate ( xylulose-5-phosphate), and finally enter the pentose phosphate pathway to p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/53C12N1/19C12P7/06C12P7/10C12R1/865C12R1/84
CPCY02E50/10
Inventor 施淑银黄德仁
Owner FAR EASTERN NEW CENTURY COPRRATION