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Multicenter monotricha xylose isomerase gene and application thereof

A xylose isomerase and gene technology, which is used in recombinant plasmids and engineering strains, and the application fields of preparing ethanol and other fermentation products

Active Publication Date: 2019-03-22
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But many yeasts that can ferment ethanol (such as Saccharomyces cerevisiae) cannot use xylose as a carbon source

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The extraction of the total DNA of multicentric monoflagellate genome of embodiment 1

[0038] ①Take about 0.5 g of the multicentric monoflagellate strain, and quickly grind it into powder in liquid nitrogen;

[0039] ②Add 4ml extract solution, shake and mix quickly;

[0040] ③Add an equal volume of 4ml of chloroform:isoamyl alcohol (24:1), vortex for 3-5min;

[0041] ④10000rpm, 4°C, 5min;

[0042] ⑤Use the volume of chloroform:isoamyl alcohol (24:1) to extract the supernatant once more (10000rpm, centrifuge at 4°C for 5min);

[0043] ⑥Take the supernatant, add 2 / 3 times the volume of -20°C pre-cooled isopropanol or 2.5 times the volume of absolute ethanol to precipitate, mix well, and let stand for about 30 minutes;

[0044] ⑦ Pick out the flocculent precipitate with a capillary glass rod, rinse repeatedly with 75% ethanol several times, then rinse once with absolute ethanol, blow dry, and resuspend in 500 μl TE;

[0045] ⑧Add 1μl RNaseA (10mg / ml), and treat at 37℃...

Embodiment 2

[0048] Example 2 Cloning of the xylose isomerase gene xylA of the multicentral monoflagellate

[0049] Using the total DNA of Monomastella multicentre extracted in Example 1 as a template, PCR amplification was performed. After the amplified product was recovered, it was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Through comparison and analysis of the sequencing sequence, it was confirmed that the obtained sequence contained the full-length gene sequence of xylose isomerase, with a total length of 1284bp. The sequence is shown in SEQ ID NO.2,

Embodiment 3

[0050] Example 3 Construction of Xylose Isomerase Gene Expression Vector Plasmid

[0051]Primers were designed using the 5' and 3' end sequences of the gene encoding Monomastella multicentricum xylose isomerase, including EcoRI and XbaI sites. The PCR product was digested with EcoRI and XbaI. The final product was cloned into a vector generated by pYES2. In this vector, the GAL1 promoter on pYES2 was replaced with the HXT1 promoter to ensure constitutive expression of xylose isomerase, thereby eliminating the need for galactose in the medium. The HXT1 promoter was cloned from the Saccharomyces cerevisiae genome. The promoter was enzymatically cleaved into the NheI-EcoRI fragment. The HXT1 promoter and the PCR product of the gene encoding xylose isomerase were ligated to pYES2 cut with EcoRI and XbaI to finally obtain the recombinant plasmid pYES-HXT-XI containing the xylose isomerase gene.

[0052] Preparation of Escherichia coli Competent Cells and Transformation of Plasm...

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PUM

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Abstract

The invention finds a novel xylose isomerase gene from multicenter monotricha. The nucleotide sequence of the novel xylose isomerase gene is shown as SEQ ID NO.2. Novel engineered yeast is obtained byintroducing recombinant plasmid containing the gene into host cells. The experiment proves that after the gene is expressed in brewer's yeast, the brewer's yeast which has no capability of convertingxylose into xylulose obtains the transformation capability. The engineered yeast can be used for preparing ethanol and other fermentation products by fermenting a culture medium containing xylose.

Description

Technical field: [0001] The present invention relates to a xylose isomerase gene, in particular to a xylose isomerase gene from a multicentric monoflagellate and its function of converting xylose into xylulose. The present invention also relates to a recombinant plasmid containing the gene and An engineering strain, and the application of the engineering strain to prepare ethanol and other fermentation products by fermenting xylose-containing medium. Background technique: [0002] The industrial production of ethanol from lignocellulose is an economical and environmentally friendly way. Because lignocellulosic raw materials from plants are renewable resources that can be obtained in large quantities. But many yeasts that can ferment ethanol (such as Saccharomyces cerevisiae) cannot use xylose as a carbon source. Therefore, there is a need to provide yeast capable of ethanol fermentation using xylose as a carbon source. [0003] Saccharomyces cerevisiae has a complete enzy...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/92C12N15/81C12N1/19C12P19/24C12P19/02C12P7/06C12R1/865
CPCC12N9/92C12N15/81C12P7/06C12P19/02C12P19/24C12Y503/01005Y02E50/10
Inventor 王智顿宝庆李桂英
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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