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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 the synthesis of N-acetylglucosamine, which can solve the problems that glucosamine cannot meet food-grade requirements and the production intensity is low.

Pending Publication Date: 2022-03-29
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0044] 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 amount of researches for utilizing microbial fermentation method to produce GlcNAc, and concrete research process is as follows:

[0045] The present invention selects the Corynebacterium glutamicum of food safety level (GARS) and is not easy to infect bacteria, and the genetic manipulation technology is mature 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 gluta...

Embodiment 1

[0152] Embodiment 1: Construction of recombinant plasmid

[0153]The primers used in this example are shown in Table 1 above.

[0154] First, the genomes of Bacillus subtilis 168 and Escherichia coli were extracted, and the glmS and yqaB genes were amplified using the genomes of Bacillus subtilis, Caenorhabditis elegans, and Escherichia coli as templates, and then the company synthesized the gene Cegna1, and these three genes were synthesized by The company carried out codon optimization, connected with the plasmid pec-xk99E through Gibson connection, and sequenced to obtain the correct plasmid pec-xk99E-BeglmS-Cegna1-yqaB.

[0155] Then use the genomes of Bacillus subtilis and Escherichia coli as templates to amplify the glmS and yqaB genes from these bacteria respectively, and then the company synthesizes the gene Cegna1, and the company optimizes the codons of these three genes, and respectively in front of the gene gna1 Add fusion tags HA, CMYC, FLag, STREPPII, and then c...

Embodiment 2

[0156] Embodiment 2: Construction of knockout plasmid

[0157] The primers used in this example are shown in Table 2 above.

[0158] Using the genome of Corynebacterium glutamicum as a template, amplify about 1000 bp of the upstream and downstream homology arms of knockout genes zwf and nanE respectively, and the obtained homology arms are zwf-L, zwf-R, nanE-L, nanE-R respectively . These homologous arms were then digested and connected to the plasmid PK-JL according to the designed restriction sites, and the correct knockout plasmids PK-JL-zwf-L-R and PK-JL-nanE-L-R were obtained by sequencing. 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 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 a phosphatase gene yqaB with the specificity of 6p-acetylglucosamine, and the genetically engineered bacterium is subjected to chassis microbial modification of related genes of a GlcNAc reverse transport pathway, a catabolism pathway and a competitive metabolic pathway which are knocked out. The genetically engineered bacterium is safe and non-toxic, can produce GlcNAc at high yield by utilizing a microbial fermentation method, and is stable in 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 C8H15NO6, molecular weight 221.21, is the product after the 2-position hydroxyl of glucose is substituted by acetylamino, is A functional monosaccharide with biological activity, it is the constituent unit of various polysaccharides in organisms, especially the exoskeleton of crustaceans has the highest content. 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, A...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/55C12P19/26C12R1/15
CPCC12N9/1096C12N9/1029C12N9/16C12N15/52C12Y203/01003C12Y206/01016C12P19/26Y02A50/30
Inventor 谭天伟刘慧李泽民
Owner BEIJING UNIV OF CHEM TECH