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Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD

A Bacillus subtilis, acetamido technology, applied in the field of genetic engineering, can solve problems such as the weakening of metabolic flow competition

Active Publication Date: 2015-12-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during ammonia synthesis, L-Gln acts as an amino donor for acetylglucosamine synthesis, and its metabolic flux is competitively weakened by L-Pro and L-Arg

Method used

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  • Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD
  • Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD
  • Method for improving acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Knockout of the gene encoding acetylglutamate semialdehyde dehydrogenase (argC), the gene encoding ornithine acetyltransferase (argJ), the gene encoding acetylglutamate kinase (argB) and the gene encoding acetylornithine transaminase (argD)

[0020] According to the upstream and downstream sequences of the gene argC encoding acetylglutamate semialdehyde dehydrogenase and the gene argD encoding acetylornithine transaminase of Bacillus subtilis (Bacillus subtilis168, purchased from the American Type Microorganism Collection, ATCC No. 27370) published on NCBI, the knockout gene was designed. Out-of-frame homologous arm amplification primers, the upstream and downstream primers of the left arm are: arg1-L-F: 5'-GCCGACCTTTCCTATGCGAAGGAC-3' and arg1-L-R: 5'-CTGTTTCCTGTGTGAAATTGTTATCCGCTCAACAAGTTCACCCTCTTGGTCTTTGTGAA-3'; the upstream and downstream primers of the right arm are: arg1 -R-F:5'-GTCGTGACTGGGAAAACCCTGGCGCTCATCATTCCGCTGTAAACCAGTGA-3' and arg1-R-R:5'-CGCGAGC...

Embodiment 2

[0021] The construction of embodiment 2 recombinant Bacillus subtilis

[0022] Transform the constructed knockout frame into Bacillus subtilis BSGN6-P xylA -glmS -P 43 -GNA1, through bleomycin resistance plate screening and colony PCR verification, it was confirmed that argCJBD was knocked out successfully, and recombinant Bacillus subtilis BSGN6ΔargCJBD::lox72-P was obtained xylA -glmS -P 43 -GNA1. Bacillus subtilis BSGN6-P xylA -glmS -P 43 - For the construction method of GNA1, please refer to the literature Liu, Y.etal.Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamineproduction.Metab.Eng.23:42-52,2014.

Embodiment 3

[0023] Example 3 Fermentative production of acetylglucosamine

[0024] The seeds cultivated at 37° C. and 200 rpm for 12 hours were transferred to the fermentation medium at an inoculum size of 5%, and cultivated at 37° C. and 200 rpm for 30 hours. After 30 hours of fermentation, the content of acetylglucosamine in the fermentation supernatant reached 6.4g / L. By knocking out argCJBD, the host bacteria are blocked from glutamic acid to arginine, which promotes the reaction of glutamic acid into glutamine, accumulates amino donors required for the synthesis of acetylglucosamine, and realizes the synthesis of acetylglucosamine. The extracellular production of recombinant Bacillus subtilis increased by 16.3% compared with that before knockout.

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Abstract

The invention discloses a method for improving the acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD, and belongs to the field of genetic engineering. Recombinant bacillus subtilis BSGN6-PxylA-glmS-P43-GNA1 serves as the original strain, acetyl glutamate glutamate-5-semialdehyde dehydrogenase coding gene (argC), ornithine acetyltransferase coding gene (argJ), acetyl glutamate kinase coding gene (argB) and acetyl ornithine aminotransferase coding gene (argD) are knocked out through homologous recombination, and therefore the approach of host bacteria for generating arginine from glutamic acid is stopped, and the reaction of converting glutamic acid into glutamine is promoted; glutamine serves as amino donors synthesized through acetylglucosamine, and therefore the yield of acetylglucosamine is improved. In the bottle shaking fermentation process through a semi-synthesized culture medium, the acetylglucosamine yield of recombinant bacillus subtilis reaches 6.4 g / L by knocking out argCJBD and is improved by 16.3% compared with the before-knockout yield. A foundation is laid for further producing glucosamine through bacillus subtilis transformed through metabolic engineering.

Description

technical field [0001] The invention relates to a method for knocking out argCJBD to increase the yield of acetylglucosamine of recombinant Bacillus subtilis, 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 enviro...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/26C12R1/125
Inventor 刘龙陈坚堵国成李江华马文龙武耀康
Owner JIANGNAN UNIV
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