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Xylitol genetic engineering producing strain as well as building method and application thereof

A genetic engineering and construction method technology, applied in the field of xylitol genetic engineering production bacteria and its construction, can solve the problems of coenzyme NADPH regeneration and strengthening to slow down the glucose utilization rate, etc. Effect

Active Publication Date: 2018-11-06
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no reports on the method of enhancing the regeneration of coenzyme NADPH and slowing down the rate of glucose utilization by blocking or reducing the flux of glucose metabolism in the EMP pathway.

Method used

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  • Xylitol genetic engineering producing strain as well as building method and application thereof
  • Xylitol genetic engineering producing strain as well as building method and application thereof
  • Xylitol genetic engineering producing strain as well as building method and application thereof

Examples

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Embodiment 1

[0053] This embodiment provides a xylitol genetically engineered production strain, which can enhance the regeneration of intracellular NADPH in an integrated xylose reductase expression strain and improve the production efficiency of xylitol.

[0054] The construction method of the xylitol genetic engineering production strain includes the following steps:

[0055] 1. Construct a pTargetF plasmid that replaces or knocks out the target gene

[0056] First, you need to find a PAM site on the target gene, that is, the NGG sequence, determine the corresponding N20 sequence, and replace the cadAspacer on the pTargetF plasmid with the N20 sequence of the target gene. Taking ptsG as an example, the plasmid construction of pTargetF-ptsG was used as an example. The primers N20-ptsG-F and N20-ptsG-R were used to perform full plasmid mutation PCR on pTargetF. The primer design methods are as follows figure 1 Shown.

[0057] The PCR recipe and program settings are as follows:

[0058] Table 1 Ful...

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Abstract

The invention discloses a xylitol genetic engineering producing strain as well as a building method and application thereof, and belongs to the technical field of genetic engineering. The xylitol genetic engineering producing strain is obtained from escherichia coli W3110 through genome modification; ptsG, xylAB and ptsF in the original escherichia coli W3110 genome are replaced into xylose reductase gene XR; in addition, at least one of pfkA, pfkB, pgi and sthA in the genome is replaced into XR. The escherichia coli W3110 is used as an initial strain; the corresponding gene in the genome is replaced into the XR; xylose reductase is efficiently expressed; meanwhile, by blocking the xylose metabolism and the xylitol phosphorylation, the xylose utilization is improved; by blocking or reducing the EMP path glucose metabolism flux, the NADPH regeneration is enhanced; necessary coenzyme NADPH is provided for reducing xylose into xylitol by xylose reductase; the efficiency of the xylitol genetic engineering producing strain for converting the xylose to produce the xylitol is greatly improved.

Description

Technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a xylitol genetic engineering production bacterium and its construction method and application. Background technique [0002] Xylitol is a five-carbon sugar alcohol. Its sweetness is equivalent to that of sucrose, but its caloric value is only about 60%. Xylitol has the characteristics of anti-caries, metabolism independent of insulin, and improvement of liver function. It is widely used in food, Pharmaceutical and chemical industry. [0003] The industrial production of xylitol mainly uses hemicellulose acid hydrolysis to obtain xylose. After separation and purification, xylose with a purity of more than 95% is obtained by catalytic hydrogenation of nickel under high temperature and high pressure conditions. The process conditions are harsh. And it is easy to cause pollution, and the production cost is high. The biological production of xylitol does not require high tempe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/18C12R1/19
CPCC12N9/0006C12N15/70C12P7/18C12Y101/01307
Inventor 吴绵斌王吉平袁新松林建平杨立荣
Owner ZHEJIANG UNIV
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