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Escherichia coli engineering bacteria and fermentation production method of n-acetylglucosamine

A technology of acetamido and Escherichia coli, applied in the field of agricultural biology, can solve the problems of energy waste, low conversion efficiency and the like

Active Publication Date: 2021-09-21
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of low conversion efficiency and waste of energy in the existing N-acetylglucosamine preparation technology, the purpose of the present invention is to provide a kind of Escherichia coli engineering bacteria producing N-acetylglucosamine

Method used

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  • Escherichia coli engineering bacteria and fermentation production method of n-acetylglucosamine
  • Escherichia coli engineering bacteria and fermentation production method of n-acetylglucosamine
  • Escherichia coli engineering bacteria and fermentation production method of n-acetylglucosamine

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Construction of Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf Chassis cells

[0038] Mutant Escherichia coli chassis cells are Escherichia coli hosts in which key genes in the glycolytic pathway and pentose phosphate pathway are knocked out, and the key gene in the glycolytic pathway that is knocked out is ATP-dependent 6-phosphofructokinase isoenzyme 1 coding gene wxya , ATP-dependent 6-phosphofructokinase isozyme 2 coding gene wxya and the coding gene of glucose-6-phosphate dehydrogenase, a key gene of pentose phosphate pathway zwf .

[0039] 1. Construction of Escherichia coli MG1655-∆ wxya strain

[0040] The pRed_cas9_recA_poxb300 plasmid and wxya Gene Targeted Knockout Vector p ∆pfkB -T3 was introduced into the E. coli MG1655 host. Using arabinose to induce the expression of Cas9 protein, cutting the genome sequence of Escherichia coli MG1655 under the guidance of gRNA, and obtaining wxya Knockout Escherichia coli MG1655-∆ wxya strain. The s...

Embodiment 2

[0066] Example 2 Construction of N-acetylglucosamine-producing E. coli MG1655- ∆pfkA∆pfkB∆zwf -pGGY engineering strain

[0067] 1. Construction of expression plasmid pGGY-1 containing genes related to N-acetylglucosamine synthesis

[0068] Will Escherichia coli Gene encoding glutamine fructose-6 phosphate transaminase glms mutant gene and Caenorhabditis legans glucosamine 6-phosphate N-acetyltransferase encoding gene gna -1 mutant gene, gene encoding fructose-1-phosphatase YqaB wxya respectively placed in P PGI Expression under the control of the promoter. The expression cassettes of each gene were assembled on the expression plasmid, and the plasmid was named pGGY-1; the specific construction method is as follows:

[0069] Amplify the nucleotide sequence of about 1800bp such as the glutamine fructose-6 phosphate transaminase gene of SEQ ID NO: 4 glms the mutant gene; Escherichia coli The MG1655 genome was used as a template for PCR to obtain a 360bp P PGI Pr...

Embodiment 3

[0076] Example 3 Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf -pGGY engineering strain fermented to produce N-acetylglucosamine

[0077] N-acetylglucosamine-producing Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf-The pGGY engineering strain was inoculated in 50 mL of LB liquid medium (ampicillin 50 μg / mL), and rejuvenated at 37°C and 200 rpm for 16 hours.

[0078] Preparation of mixed carbon source fermentation medium: Accurately weigh 6.78 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 1.5 g of sodium chloride, 1 g of ammonium chloride, 10 g of glucose, 0.011 g of calcium chloride, and sulfuric acid heptahydrate Magnesium 0.493 g, glycerol 5 g, yeast extract 0.5 g, peptone 1 g, water as a solvent to make up to 1 L, 115 ℃ autoclaving for 30 minutes to obtain a mixed carbon source fermentation medium.

[0079] Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf -The pGGY engineering strain was transferred to 200mL mixed fermentation medium (ampicillin 50 μg / mL) accordi...

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Abstract

The application relates to the field of agricultural biotechnology, in particular to an Escherichia coli engineering bacterium and a fermentation production method of N-acetylglucosamine. The present invention improves the supply of product precursors in mutant Escherichia coli chassis cells by cutting off the shunting of N-acetylglucosamine synthesis precursor 6-fructose phosphate by truncating the glycolysis pathway and the pentose phosphate pathway, and then utilizing the free high-copy The plasmid expresses the key enzymes of the N-acetylglucosamine synthesis pathway in the chassis cells, and the mixed carbon source medium is used to ferment and produce N-acetylglucosamine, and the yield of N-acetylglucosamine reaches 2.8 g / L.

Description

technical field [0001] The application relates to the field of agricultural biotechnology, in particular to an Escherichia coli engineering bacterium of N-acetylglucosamine and a fermentation production method. Background technique [0002] Glucosamine is a compound in which the hydroxyl groups of glucose molecules are replaced by amino groups. Usually in the form of N-acetyl derivatives or N-sulfate esters and N-acetyl-3-O-lactate ethers in cell wall-bound polysaccharides and animal connective tissues. Industrially produced glucosamine has been widely used in feed, medicine, food, daily chemical and many other industries. Among them, N-acetylglucosamine can repair damaged chondrocyte tissue in the human body, increase the lubrication between joints, and plays an important role in the field of medicine, especially in the treatment and prevention of arthritis. [0003] At present, most of the N-acetylglucosamine in my country uses chitosan as raw material and is produced th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/55C12P19/26C12P19/02C12R1/19
CPCC12N9/0006C12N9/1029C12N9/1096C12N9/1205C12N9/16C12P19/02C12P19/26C12Y101/01049C12Y203/01004C12Y206/01016C12Y207/01011C12Y207/0103
Inventor 张杰王凯凯王晓璐姚斌罗会颖黄火清苏小运柏映国涂涛王苑王亚茹秦星
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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