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Regulation and control method for producing xylose by transforming xylan by use of escherichia coli

A technology of Escherichia coli and xylan, which is applied in the field of the construction of xylose engineering strains by transforming xylan to produce xylose and the regulation of pathways, can solve the problems of complex process and high cost, and achieves simplified experimental steps, simplified production process, and reduced production costs. Effect

Inactive Publication Date: 2015-04-29
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for producing xylose by transforming xylan with E. Construction and Control Methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Selection of regulatory element promoters

[0013] 1. Clone the DNA fragments of the constitutive promoters gapA, pgi, pykA and pykF from E. coli C600;

[0014] 2. Cloning from the plasmid T-vector-eGFP as a reporter gene for characterizing the promoter strength—enhanced green fluorescent protein (eGFP) gene fragment;

[0015] 3. The DNA junction fragments gapA-eGFP, pgi-eGFP, pykA-eGFP and pykF-eGFP of eGFP and promoter were respectively amplified by overlapping extension PCR;

[0016] 4. Use BamH I and Bgl II to double-digest the DNA junction fragment and plasmid pET-28a(+), respectively, use T4 DNA ligase to connect each DNA junction fragment with the linear plasmid pET-28a(+), and transform Escherichia coli BL21(DE3), four recombinant engineering bacteria were obtained;

[0017] 5. Measure the fluorescence intensity of promoters gapA, pykF, pgi and pykA by flow cytometry, and use them as regulatory elements in the construction of pathways for converting...

Embodiment 2

[0018] Embodiment 2: the construction of escherichia coli genetic engineering bacterium

[0019] 1. Clone the xylanase Xyn and xylosidase Xyl gene fragments from Aspergillus terreus Li-20, and find that they are respectively identical to the A. terreus Li-20 xylanase gene sequence published in Genbank (Genbank registered The sequence number is JQ087496) and the xylosidase gene sequence (Genbank registration sequence number is JQ087497) is 100% homologous;

[0020] 2. Clone the gene fragment of the carrier protein OsmY and the DNA fragment of the terminator rrnb from Escherichia coli BL21 (DE3);

[0021] 3. Clone the DNA fragment of the constitutive promoter from Escherichia coli C600;

[0022] 4. Clone the DNA fragment of the terminator T7 from the plasmid pET-28a(+);

[0023] 5. Construct the xylanase gene expression cassette and the xylosidase gene expression cassette by overlapping extension PCR, and transfer them into Saccharomyces cerevisiae INVSc1 by electroporation to...

Embodiment 3

[0024] Embodiment 3: the fermentation experiment of engineering bacterial strain

[0025] The engineered bacteria seed solution was initially OD 550 The amount of 0.1 was inserted into the LB medium with the initial xylan concentration of 3g / L, and the fermentation was carried out at 37°C and 170rpm for 96h. Centrifuge the fermented liquid samples taken at different time points, take the supernatant, and filter it through a water phase filter membrane with a pore size of 0.22 μm. The highest yield of xylose is 90.19 mg / L as detected by HPLC.

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Abstract

The invention provides a regulation and control method for producing xylose by transforming xylan by use of escherichia coli and belongs to the field of biochemical engineering. According to the regulation and control method, the constitutive promoters gapA, pgi, pykA and pykE of the escherichia coli are cloned and characterized, and applied to constructing an approach of producing the xylose by transforming the xylan; and the expression of OsmY-Xyn (the fusion protein of carrier protein and xylanase) and the expression of OsmY-Xyl (the fusion protein of carrier protein and xylosidase) are controlled, respectively, and the transcription of the two fusion proteins is terminated by use of terminators rrnb and T7, respectively. The approach is imported into the escherichia coli BL21 (DE3) in the form of plasmids to obtain a genetic engineering strain BL21-Xyn-XYl capable of producing the xylose from the xylan. The method of regulating and controlling the approach of producing the xylose by transforming the xylan by use of the escherichia coli by adopting the characterized constitutive promoters is adopted, the addition of an inducer and the optimization of induction conditions are avoided, and then the production process can be simplified and the production cost can be reduced.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to the construction of engineering strains for xylan conversion to produce xylose and the regulation method of the pathway. Background technique [0002] Xylan is the most abundant polysaccharide in nature. It is the main component of hemicellulose in plant cells, accounting for 35% of the dry weight of plant cells. Xylooligosaccharides can be obtained by partially degrading xylan, and five-carbon monosaccharides (xylose, ferulose, arabinose, etc.) can be obtained by complete degradation, among which xylose accounts for the largest proportion. [0003] Xylose is an important chemical raw material, which is widely used in food, medicine, chemical industry, leather, dyes and other fields. Xylose can activate bifidobacteria (beneficial bacteria, beneficial to human health) in the human intestinal tract and promote their growth, improve the microbial environment of the human body, and...

Claims

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Application Information

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IPC IPC(8): C12P19/02C12N15/70C12R1/19
Inventor 李春王翠薇张根林李哲
Owner SHIHEZI UNIVERSITY
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