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Genetically engineered bacterium for synthesizing N-acetylglucosamine and application of genetically engineered bacterium

A technology of genetically engineered bacteria and glucosamine, which is applied in the field of genetically engineered bacteria for synthesizing N-acetylglucosamine, and can solve the problems of low production intensity and the inability of glucosamine to meet food-grade requirements.

Active Publication Date: 2020-01-10
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As early as 2005, MingDe Deng et al. used Escherichia coli to produce 110g / LGlcNAc through metabolic engineering and fermentation process optimization. However, because E. coli would secrete endotoxin during the fermentation process, the glucosamine produced could not meet the requirements of food grade. need
In recent years, the production of GlcNAc by yeast fermentation has been reported, the highest yield is only 3g / L, and the fermentation cycle is as long as 135 hours, so the production intensity is low

Method used

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  • Genetically engineered bacterium for synthesizing N-acetylglucosamine and application of genetically engineered bacterium
  • Genetically engineered bacterium for synthesizing N-acetylglucosamine and application of genetically engineered bacterium
  • Genetically engineered bacterium for synthesizing N-acetylglucosamine and application of genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0042] In the existing technology of using microbial fermentation to produce GlcNAc, Escherichia coli will secrete endotoxin during the fermentation process, and the glucosamine produced cannot meet the food-grade requirements; the highest yield of GlcNAc produced by yeast fermentation is only 3g / L, and The fermentation cycle is as long as 135 hours, and the production intensity is low. In view of this, the present inventor has carried out a large number of studies on the production of GlcNAc by microbial fermentation, and the specific research process is as follows:

[0043] The present invention selects food safety grade (GARS) Corynebacterium glutamicum which is not easy to infect bacteria and has mature gene manipulation technology to produce GlcNAc. The production rate of GlcNAc is directly related to the growth rate of bacteria, and it is a primary metabolite, so the fermentation type of GlcNAc belongs to the growth-coupled type. Corynebacterium glutamicum can use gluco...

Embodiment 1

[0126] Embodiment 1: Construction of recombinant plasmid

[0127] The primers used in this example are shown in Table 1 above.

[0128] Firstly, the genomes of Corynebacterium glutamicum, Saccharomyces cerevisiae, and Escherichia coli were extracted, and the glmS, gna1, and yqaB genes were amplified using the genomes of Corynebacterium glutamicum, Saccharomyces cerevisiae, and Escherichia coli as templates, and these three genes were sequentially combined with the plasmid pec-xk99E was connected, and the correct plasmids pec-xk99E-cglglmS, pec-xk99E-gna1 and pec-xk99E-cglglmS-gna1, pec-xk99E-cglglmS-gna1-yqaB were obtained by sequencing.

[0129] Then, the glmS genes from Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis were amplified as templates. The glmS genes from different sources were digested and ligated with the plasmid pec-xk99E-gna1, and sequenced to obtain the correct plasmids pec-xk99E-EcglmS-gna1-yqaB, pec-xk99E-ScglmS-gna1-yqaB, pec-xk99E-BsglmS...

Embodiment 2

[0130] Embodiment 2: Construction of knockout plasmid

[0131] The primers used in this example are shown in Table 2 above.

[0132] Using the genome of Corynebacterium glutamicum as a template, amplify about 1000bp of the upstream and downstream homology arms of knockout genes nagA / B, ldh, and cgl2642, and the resulting homology arms are nagA / B-L, nagA / B-R, and ldh-L, respectively , ldh-R, cgl2642-L, cgl2642-R. Then these homology arms were digested and ligated with the plasmid PK-JL according to the designed restriction sites, and sequenced to obtain the correct knockout plasmids PK-JL-nagA / B-L-R, PK-JL-ldh-L-R, PK-JL -cgl2642-L-R. Subsequently, each gene was knocked out according to the traditional knockout method of grain stick gene.

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Abstract

The invention relates to a genetically engineered bacterium for synthesizing N-acetylglucosamine and an application of the genetically engineered bacterium. The genetically engineered bacterium is recombinant corynebacterium glutamicum containing a glucosamine synthetase gene glmS and a glucosamine transacetylase gene gna1, and is modified by a chassis microorganism of which related genes of a GlcNAc reverse transport pathway, a catabolism pathway and a competitive metabolism pathway are knocked out; the genetically engineered bacterium also contains a 6p-GlcNAc specific phosphatase gene yqaBused for increasing the concentration of extracellular GlcNAc, and the genetically engineered bacterium expresses the glucosamine synthetase gene glmS, the glucosamine transacetylase gene gna1 and the6p-GlcNAc specific phosphatase gene yqaB. The genetically engineered bacterium is safe and non-toxic, can produce the GlcNAc at high yield by utilizing a microbial fermentation method, and is stablein batch, relatively low in production cost and wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and relates to a genetically engineered bacterium for synthesizing N-acetylglucosamine and its application. Background technique [0002] N-acetylglucosamine, also known as N-acetylglucosamine (N-acetyl-D-(+)-glucosamine, GlcNAc), molecular formula C 8 h 15 NO 6 , with a molecular weight of 221.21, is the product of the 2-hydroxyl group of glucose being replaced by an acetamido group. It is a functional monosaccharide with biological activity and is the constituent unit of various polysaccharides in organisms, especially in the exoskeleton of crustaceans. GlcNAc is an important precursor for the synthesis of bifidus factors, and has many important physiological functions in vivo; it has cartilage damage repair and regeneration functions; it can also improve immune regulation, stimulate anti-tumor immune response, and has anti-tumor and anti-inflammatory functions; at the same time, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/28C12R1/15
CPCC12N9/1029C12N9/1096C12P19/28C12Y206/01016
Inventor 谭天伟王秋婷郑昭奕王梦
Owner BEIJING UNIV OF CHEM TECH
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