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Genetic engineering strain, construction method and application in xylitol production

A genetically engineered bacteria and genetic technology, applied in the field of genetic engineering and biology, can solve the problems of xylose reductase catalytic efficiency, cell growth rate reduction, etc., to achieve the effects of increased production rate, recovery of growth defects, and reduced utilization rate

Active Publication Date: 2015-05-06
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the conventional culture temperature of Escherichia coli is 37°C. Fermentation at such a low temperature will reduce the cell growth rate, and the catalytic efficiency of xylose reductase will also be affected by low temperature.

Method used

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  • Genetic engineering strain, construction method and application in xylitol production
  • Genetic engineering strain, construction method and application in xylitol production
  • Genetic engineering strain, construction method and application in xylitol production

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Experimental program
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Effect test

Embodiment 2

[0185] Embodiment 2 utilizes the example of engineering bacterium HK402 to produce xylitol

[0186] 1. Engineering bacteria use corncob hemicellulose hydrolyzate to produce xylitol

[0187] In order to verify whether the constructed engineering bacteria can utilize hemicellulose hydrolysis to produce xylitol, the engineering bacteria HK402 was used to carry out shake flask fermentation (250ml, liquid volume is 50ml).

[0188] (1) Inoculate the overnight seed medium with engineering bacteria HK402 at 2%, and cultivate it at 30°C for 8 hours to obtain the seed solution;

[0189] The formula of seed medium and fermentation medium is: 1L of medium contains 4-6g Na 2 HPO 4 ,2~5g KH 2 PO 4 ,1~2g NH 4 Cl, 1~5g NaCl, 1~5mM MgSO 4 ,1~5mM CaCl 2 , 2-10g / L yeast extract.

[0190] (2) Concentrate the hemicellulose hydrolyzate with a rotary evaporator until the xylose content is 20%-50%, sterilize the concentrated hemicellulose hydrolyzate and add it to the sterilized fermentation ...

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Abstract

The invention discloses a genetic engineering strain, a construction method and application thereof in xylitol production. The genetic engineering strain includes Escherichia coli and an expression vector transferred into the Escherichia coli. An XR gene is inserted to the downstream of the expression vector's promoter, the base sequence of the XR gene is shown as SEQ ID No.4, the promoter is Trc promoter, the 5' end of the XR gene has RBS from pET30a, and at least one of ptsG, xylA, xylB and ptsF genes in the Escherichia coli is inhibited or knocked out. Through translation initiation rate screening on xylose reductase gene and selection of appropriate RBS and promoter, the XR gene in the genetic engineering strain can obtain high efficiency expression at 30DEG C, and no inclusion body is generated. By knockout of the ptsG gene, the glucose effect is eliminated, and through knockout of the xylA, xylB and ptsF genes, xylose metabolism and xylitol phosphorylation are blocked.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, in particular to a genetically engineered bacterium, its construction method and its application in producing xylitol Background technique [0002] At present, the industrial production of xylitol is mainly produced by hydrogenation of xylose with nickel catalysis under high temperature and high pressure conditions. This process requires harsh conditions and is easy to cause pollution. Therefore, the production of xylitol by biological conversion is more and more popular. Pay attention to. The microorganisms used to prepare xylitol by fermentation are almost all yeasts, including natural strains and genetically engineered strains. Yeast has its own advantages as a xylitol producing strain, such as being able to tolerate higher sugar concentrations, and having stronger resistance to inhibitory factors in hemicellulose hydrolyzate, and so on. But there are also unavoidable pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/18C12R1/19
CPCC12N9/0006C12P7/18C12Y101/01307
Inventor 吴绵斌苏卜利张哲林建平杨立荣
Owner ZHEJIANG UNIV
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