A high-yielding glucosamine engineering bacterium and its construction method through homologous recombination knockout of manx

A technology of glucosamine and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of low conversion efficiency of chitin hydrolysis, not yet reached industrialized production, and high product cost, and achieve low production cost, high production intensity, and low environmental pollution. Effect

Active Publication Date: 2011-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Chitin hydrolysis is currently the main method for producing glucosamine in my country. The conversion efficiency of chitin hydrolysis is low, and a large amount of acidic wastewater is produced, so the product cost is high and the pollution is serious.
At present, there are few reports on the production of glucosamine by biological methods at home and abroad, and the fermentation yield is also low, which has not yet reached the requirements of industrial production.

Method used

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  • A high-yielding glucosamine engineering bacterium and its construction method through homologous recombination knockout of manx
  • A high-yielding glucosamine engineering bacterium and its construction method through homologous recombination knockout of manx
  • A high-yielding glucosamine engineering bacterium and its construction method through homologous recombination knockout of manx

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Experimental program
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Effect test

Embodiment Construction

[0015] 1. Construction of recombinant plasmid pET-28a(+)-glmS-gna1

[0016] Primers used to amplify the glmS gene:

[0017] Upstream: 5'-C GA GCT C AT GTG TGG AAT TGT TGG C-3' (underlined sequence indicates restriction endonuclease recognition site Sac I)

[0018] Downstream: 5'-CCC AAG CTT TTA CTC AAC CGT AAC CGA-3' (the underlined sequence represents the restriction enzyme site Hind III).

[0019] Glucosamine synthase gene glmS was obtained by PCR using E. coli BL21 (DE3) genome (GenBank No. CP001509.3) as a template. Digest the glmS gene fragment and pET-28a(+) with restriction endonucleases Sac I and Hind III, and insert the glmS gene fragment into the Sac I and Hind III sites of plasmid pET-28a(+) by ligation reaction In between, the recombinant plasmid pET-28a(+)-glmS was obtained.

[0020] Primers used to amplify the gna1 gene:

[0021] Upstream: 5'-AAGGAGATAAGAAT GCGGCCGC ATGAGCTTAC-3' (underlined sequence indicates restriction endonuclease recognition site No...

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Abstract

The invention discloses a high-glucosamine-yield engineering bacterium and use thereof. In the invention, the gene engineering bacterium E.coli-glmS-gnal-deltanagE-deltamanX is obtained by transferring a glucosamine synthase gene (glmS) and a glucosamine acetyltransferase (gnal) into E.coli K-12 and knocking out an acetylglucosamine phosphoric acid transfer system nagE gene and a mannose phosphoric acid transfer system manX gene; the strain is used for fermenting to produce glucosamine, and has the advantages of short fermentation time, high production strength, low production cost, small environment pollution, no allergic reaction and the like; and the produced glucosamine can be widely used in fields of medicines, foods and the like.

Description

technical field [0001] The invention relates to a glucosamine-producing genetically engineered bacterium and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Glucosamine (Glucosamine, 2-amino-2-deoxy-D-glucose) is a compound in which a hydroxyl group of glucose is replaced by an amino group. It exists in almost all organisms, including bacteria, yeast, filamentous fungi, plants and animals, and is the main component of glycoproteins and proteoglycans. Glucosamine can specifically act on articular cartilage, restore the normal metabolic function of chondrocytes, stimulate chondrocytes to produce proteoglycans with normal polymer structure, inhibit enzymes that damage cartilage, and delay the pathological process and disease of osteoarthritis progression, improved joint mobility, and pain relief. Therefore, it is mostly used clinically for the treatment of osteoarthritis. In addition, glucosamine also has liver...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/60C12N15/63C12N15/09C12R1/19
Inventor 陈坚堵国成刘龙李江华陈欣何菊
Owner JIANGNAN UNIV
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