A method for knocking out argcjbd to improve the production of acetylglucosamine in recombinant Bacillus subtilis

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

Active Publication Date: 2017-12-12
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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  • A method for knocking out argcjbd to improve the production of acetylglucosamine in recombinant Bacillus subtilis
  • A method for knocking out argcjbd to improve the production of acetylglucosamine in recombinant Bacillus subtilis
  • A method for knocking out argcjbd to improve the production of acetylglucosamine in recombinant Bacillus subtilis

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 acetylglutamate semialdehyde dehydrogenase encoding gene argC and the acetylornithine aminotransferase encoding gene argD of Bacillus subtilis 168 (purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI , design primers for homology arm amplification of the knockout frame, 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 respectively For: arg1-R-F:5'-GTCGTGACTGGGAAAACCCTGGCGCTCATCATTCCGCTGTAAACCAGTGA-3' and arg1-R-R:5'-CGCGAGCGCGATCAGCTGA...

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. et al. Modular pathway engineering of Bacillus subtilis forimproved N-acetylglucosamine production. 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 knocking out argCJBD to increase the yield of acetylglucosamine of recombinant Bacillus subtilis, belonging to the field of genetic engineering. The invention uses the recombinant Bacillus subtilis BSGN6-PxylA-glmS-P43-GNA1 as the starting strain, and knocks out the gene encoding acetylglutamate semialdehyde dehydrogenase (argC) and the gene encoding ornithine acetyltransferase through homologous recombination (argJ), acetylglutamate kinase encoding gene (argB) and acetylornithine transaminase encoding gene (argD), block the pathway of host bacteria producing arginine from glutamic acid, and promote the conversion of glutamic acid into glutamic acid The reaction of amide, and glutamine is the amino donor for the synthesis of acetylglucosamine, thereby increasing the production of acetylglucosamine. During the shake flask fermentation process using semi-synthetic medium, the yield of acetylglucosamine of the recombinant Bacillus subtilis with knockout of argCJBD reached 6.4 g / L, which was 16.3% higher than that before knockout. It laid the foundation for further metabolic engineering of Bacillus subtilis to produce glucosamine.

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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Patent Type & Authority Patents(China)
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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