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Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis

A technology of Bacillus subtilis and glucosamine, applied in the field of genetic engineering, can solve the problem of reducing the concentration of extracellular acetylglucosamine, and achieve the effect of simple construction method, good application prospect and easy use

Active Publication Date: 2014-01-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, acetylglucosamine is a carbon source preferentially used by Bacillus subtilis. When the glucose in the medium is depleted, extracellular acetylglucosamine is transported into the cell and decomposed and utilized, resulting in a rapid increase in the concentration of extracellular acetylglucosamine. reduce

Method used

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  • Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis
  • Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis
  • Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Knockout of the gene encoding acetylglucosamine transporter (nagP)

[0018] According to the upstream and downstream sequences of the acetylglucosamine transporter gene (nagP) of Bacillus subtilis (Bacillus subtilis 168, purchased from the American Type Microorganism Collection, ATCC No. 27370) published on NCBI, primers were designed to amplify the homology arms of the knockout frame, The upstream and downstream primers of the left arm are: nagP-L-F: 5'-AATGAGATGCCTGTGTCGGAATA-3' and nagP-L-R:

[0019] 5'-TCCTGTGTGAAATTGTTATCCGCTCATCCACTCTCCAAACGAGTTGATAC-3'; the upper and lower primers of the right arm are: nagP-R-F:

[0020] 5'-ACGTCGTGACTGGGAAAACCCTGGCCGCGGTCTTAACCGGGTTA-3' and nagP-R-R:

[0021] 5'-TACGACAACGCCCAGCTTC-3'. The left and right arms contained in the knockout frame were amplified from the Bacillus subtilis 168 genome using the above primers. According to the p7Z6 plasmid sequence published on NCBI (gifted by Dr. Yan Xin, Nanjing Agricultura...

Embodiment 2

[0024] The construction of embodiment 2 recombinant Bacillus subtilis

[0025] The constructed knockout frame was transformed into Bacillus subtilis (Bacillus subtilis 168), and through bleomycin resistance plate screening and colony PCR verification, it was confirmed that the gene encoding acetylglucosamine transporter (nagP) was successfully knocked out, and recombinant Bacillus subtilis was obtained Bacillus BSGN1.

[0026] According to the glucosamine acetylase encoding gene (GNA1) in Saccharomyces cerevisiae S288C (purchased from the American Type Microorganism Collection, No. ATCC204508) published on NCBI, the primer GNA1-F was designed: 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA1 -R: 5'-CCCAAGCTTCTATTTTTCTAATTTGCATTTCCACG-3'. The gene encoding glucosamine acetylase (GNA1) was amplified from the Saccharomyces cerevisiae S288C genome using the above primers. The amplified fragment was digested with KpnI and HindIII and then ligated into the p...

Embodiment 3

[0028] Example 3 Fermentative production of acetylglucosamine

[0029] The seeds cultivated at 37°C and 200rpm for 12h were transferred to the fermentation medium with an inoculum size of 5%, and cultivated at 37°C and 200rpm for 30h. After 30 hours of fermentation, the content of acetylglucosamine in the fermentation supernatant reached 415 mg / L. Increased extracellular production of acetylglucosamine in recombinant Bacillus subtilis was achieved by knocking out the gene encoding the acetylglucosamine transporter (nagP) and overexpressing the gene encoding glucosamine acetylase (GNA1) in a nagP-knockout host .

[0030]

[0031]

[0032]

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Abstract

The invention discloses a recombination bacillus subtilis with high yield of acetylglucosamine, and an application of the recombination bacillus subtilis, which belong to the field of genetic engineering. According to invention, bacillus subtilis 168 is taken as an original strain, a translocator encoding gene (nagP) of the acetylglucosamine is knocked out by virtue of homologous recombination, and thus a path for host bacteria to transport the acetylglucosamine from an extracellular part to an intracellular part is blocked. In the host bacteria the nagP of which is knocked out, over-expression is derived from glucosamine acetylase encoding gene (GNA1) of saccharomyces cerevisiae S288C, so that the synthesis path of the acetylglucosamine is improved, and the yield of acetylglucosamine in the recombination bacillus subtilis is enhanced to reach 415mg / L. Therefore, the application lays a foundation for producing glucosamine by improving the bacillus subtilis through metabolic engineering.

Description

technical field [0001] The invention relates to a high-production acetylglucosamine recombinant bacillus subtilis and its application, belonging to the field of genetic engineering. Background technique [0002] Acetyl glucosamine is a kind of monosaccharide in organisms, which widely exists in bacteria, yeast, mold, plants and animals. In the human body, acetylglucosamine is the synthetic precursor of the disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of cartilage and joint tissue functions. Therefore, acetyl glucosamine is widely used as a drug and nutritional dietary supplement to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the field of cosmetics and pharmaceuticals. At present, acetyl glucosamine is mainly produced by acid-decomposing chitin in shrimp shells or crab shells. The waste liquid produced by this method is relatively serious for environmental pollution, and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/09C12N15/55C12N15/75C12P19/26C12R1/125
Inventor 陈坚堵国成刘龙李江华刘延峰
Owner JIANGNAN UNIV