A kind of genetically engineered bacteria producing n-acetylglucosamine and its application

A technology of genetically engineered bacteria and acetylamino, which is applied in the field of genetically engineered bacteria producing N-acetylglucosamine, can solve the problems of strong acid waste liquid polluting the environment, strong hydrolysis conditions, and crowd allergies

Active Publication Date: 2021-01-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has disadvantages such as strong acid waste liquid polluting the environment, strong hydrolysis conditions, and the source of shrimp shells and crab shells causing allergies in some people.

Method used

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  • A kind of genetically engineered bacteria producing n-acetylglucosamine and its application
  • A kind of genetically engineered bacteria producing n-acetylglucosamine and its application
  • A kind of genetically engineered bacteria producing n-acetylglucosamine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Construction of E.coli W3110-GLA-1 strain

[0109] (1) Knockout of nagBAC gene cluster:

[0110] Using CRISPR / Cas9 gene editing technology to knock out the nagBAC gene cluster:

[0111] ①Using PCR technology to use the E.coli W3110 genome as a template, according to the nagBAC gene sequence, design upstream homology arm primers (nagBAC-UF / UR) and downstream homology arm primers (nagBAC-DF / DR) at both ends of the gene, PCR Amplify and obtain the upstream and downstream homology arms of the nagBAC gene;

[0112] ②Using overlapping PCR technology to use the upstream and downstream homology arms of the nagBAC gene as templates, PCR amplification was used to obtain the overlapping fragments of the upstream and downstream homology arms of the nagBAC gene;

[0113] ③ Construct a gRNA plasmid containing the Cas9 cleavage recognition sequence. The DNA fragment containing the target sequence is obtained by annealing the primers nagBAC-F and nagBAC-R. The constructed gRNA plasmi...

Embodiment 2E

[0161] Embodiment 2E.coli W3110-GLA-1 bacterial strain 5L fermentation tank fermentation experiment:

[0162] The E.coli W3110-GLA-1 strain constructed in Example 1 is used as a production strain to ferment and produce N-acetylglucosamine:

[0163] (1) Slope activation culture: Scrape a ring of bacteria from the -80°C refrigerator bacteria preservation tube, evenly spread on the activation slope, culture at 37°C for 12 hours, transfer to the second generation slope culture for 12 hours;

[0164] Seed culture: Aseptic operation, take appropriate amount of sterile water on the second-generation activation slope, put the bacterial suspension into a 5L fermenter with 3L seed medium, and control the automatic flow of ammonia water through the pH electrode to maintain the pH during the cultivation process Stable at around 7.0; the temperature of the fermentation process is automatically controlled at 37°C by the temperature electrode; the dissolved oxygen is controlled between 25-35...

Embodiment 3E

[0171] Embodiment 3E.coli W3110-GLA-2 bacterial strain 5L fermentation tank fermentation experiment:

[0172] Use the constructed E.coli W3110-GLA-2 strain as the production strain to ferment and produce N-acetylglucosamine:

[0173] (1) Slope activation culture: Scrape a ring of bacteria from the -80°C refrigerator bacteria preservation tube, evenly spread on the activation slope, culture at 37°C for 12 hours, transfer to the second generation slope culture for 12 hours;

[0174] Seed culture: Aseptic operation, take appropriate amount of sterile water on the second-generation activation slope, put the bacterial suspension into a 5L fermenter with 3L seed medium, and control the automatic flow of ammonia water through the pH electrode to maintain the pH during the cultivation process Stable at around 7.0; the temperature of the fermentation process is automatically controlled at 37°C by the temperature electrode; the dissolved oxygen is controlled between 25-35% by the rotati...

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PUM

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Abstract

The invention discloses a genetically engineered bacterium that produces N-acetylglucosamine and an application thereof. The genetically engineered bacterium uses arabinose or rhamnose to induce simultaneously enhancement of N-acetylglucosamine synthesis pathways and weakening and weakening of bypassing metabolic pathways to achieve accurate control. The N-acetylglucosamine can be efficiently synthesized from constructed Escherichia coli genetically engineered bacterium W3110-GLA-1 (arabinose inducible type) and W3110-GLA-2 (rhamnose inducible type) with glucose as a substrate, the yield of the N-acetylglucosamine can reach 168g / L and 160g / L after 72 hours fermentation in a 5L fermentation tank, the conversion rate is about 48% and 47%, and high industrial production potential is achieved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetically engineered bacterium producing N-acetylglucosamine and its application. Background technique [0002] N-Acetyl-D-Glucosamine (N-Acetyl-D-Glucosamine) is a derivative of glucose whose hydroxyl group is replaced by an amino group, also known as N-acetyl-D-glucosamine, and its chemical name is 2-acetylamino-2-deoxy -D-glucose. N-acetylglucosamine can be deacetylated and converted into glucosamine under acidic conditions. At present, glucosamine is widely used in medicine, food, cosmetics and other fields, and has a large market demand. [0003] Glucosamine is used in the clinical treatment of arthritis, osteoarthritis, rheumatoid arthritis, cartilage damage, joint damage and other diseases. In addition, N-acetylglucosamine can inhibit the activity of elastase and inhibit the release of superoxide from human polymorphonuclear leukocytes. In addition, N-a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/19
CPCC07K14/245C12N9/1096C12N9/1205C12N9/1288C12P19/26C12Y207/01011
Inventor 马倩谢希贤陈宁张权威鄢芳清侯正杰莫晓琳徐庆阳李燕军张成林范晓光
Owner TIANJIN UNIV OF SCI & TECH
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