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A method for promoting the synthesis of acetylglucosamine in Bacillus subtilis

A technology of Bacillus subtilis and recombinant bacteria, which is applied in the field of genetic engineering, can solve the problems of metabolic overflow and NADH consumption, and achieve the effect of simple construction method, reduction of NADH, and balance of intracellular reducing power

Active Publication Date: 2020-05-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the cells need to maintain the balance of reducing power, the excessively produced NADH will be consumed in two ways, one is involved in the synthesis of other metabolisms (leading to the production of by-products), and the other is oxidized to produce ATP (resulting in the consumption of a large amount of O 2 , it will also cause a large amount of bacteria to be produced)
In addition, the synthesis of acetylglucosamine by Bacillus subtilis is accompanied by metabolic overflow, resulting in a large synthesis of the by-product acetoin

Method used

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  • A method for promoting the synthesis of acetylglucosamine in Bacillus subtilis
  • A method for promoting the synthesis of acetylglucosamine in Bacillus subtilis
  • A method for promoting the synthesis of acetylglucosamine in Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of Bacillus subtilis BSGNKAP3

[0049] Bacillus subtilis BSGNKAP2 for B. subtilis 168ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔptaΔgl cKΔpckAΔpyk P 43 -glmS P 43 - pycA::lox72, episomal expression of the GNA1 gene from the pP43NMK-GNA1 plasmid. Then, on this basis, the gene balpycA encoding pyruvate carboxylase BalpycA (NCBI-ProteinID: AAS42897) of Bacillus cereus was integrated at the malS site of the Bacillus subtilis genome, and was verified by bleomycin resistance plate screening and colony PCR , Sequencing, confirming that the integration was successful to obtain the recombinant Bacillus subtilis BSGNKAP3.

Embodiment 2

[0050] Example 2: Construction of Bacillus subtilis BSGNKAP4

[0051] Bacillus subtilis BSGNKAP3 was used as the host, and the pP43NMK-GNA1 plasmid expressed the GNA1 gene freely, and then integrated glyceraldehyde-3-phosphate ferredoxin dehydrogenase (NCBI-ProteinID: CAF30501 ) encoding gene gor, through bleomycin resistance plate screening, colony PCR verification, and sequencing, it was confirmed that the integration was successful and the recombinant Bacillus subtilis BSGNKAP4 was obtained.

Embodiment 3

[0052] Example 3: Construction of recombinant Bacillus subtilis BSGNKAP5

[0053] Using BSGNKAP4 as the host, the GNA1 gene was freely expressed with the pP43NMK-GNA1 plasmid, and then integrated isocitrate NAD at the ywkA site of the Bacillus subtilis genome + Dehydrogenase (NCBI-Protein ID: AKC61181) encoding gene icd was successfully integrated to obtain recombinant Bacillus subtilis BSGNKAP5 through bleomycin resistance plate screening, colony PCR verification, and sequencing.

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Abstract

The invention discloses a method for promoting the synthesis of acetylglucosamine of bacillus subtilis, which belongs to the field of genetic engineering. The present invention uses the recombinant Bacillus subtilis BSGNKAP2 as the starting strain, and exogenously introduces pyruvate carboxylase BalpycA derived from Bacillus cereus to eliminate the overflow of carbon metabolism in the center of Bacillus subtilis, avoid the synthesis of by-product acetoin, and further Introduce 5 exogenous reducing power metabolism reactions to replace the glycolysis pathway, the reaction of NADH in the tricarboxylic acid cycle, and reconstruct the intracellular reducing power metabolism, including glyceraldehyde-3-phosphate ferredoxin dehydrogenase, iso CitrateNAD + Dehydrogenase, malate quinone dehydrogenase, ketoferredoxin oxidoreductase, and nitrogenase ferritin. During the shake flask fermentation using the compound medium, the yield of acetylglucosamine of the recombinant strain BSGNKAP8 was 24.50g / L, and the yield of acetylglucosamine / glucose was 0.469g / g, which were 1.97 and 2.13 times that of the starting strain BSGNKAP2, respectively.

Description

technical field [0001] The invention relates to a method for promoting the synthesis of acetylglucosamine of bacillus subtilis, belonging to the field of genetic engineering. Background technique [0002] 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 added in medicine and nutritional diet to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the field of cosmetics and pharmaceuticals. At present, acetylglucosamine is mainly produced by acid-decomposing chitin in shrimp shells or crab shells. However, the waste liquid produced by this method is relatively serious for environmental pollution, and the obtained products are likely to cause allergic reactions, so it is not suitable for people with seafood allergies. [0003] Bacillus s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/52C12P19/26C12R1/125
CPCC12N9/0006C12N9/0008C12N9/0095C12N9/93C12P19/26C12Y101/01037C12Y101/01041C12Y102/07001C12Y102/07006C12Y118/06001C12Y604/01001C12Y101/05004C12N15/52
Inventor 刘龙顾洋邓洁莹陈坚堵国成李江华
Owner JIANGNAN UNIV
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