Method for promoting synthesis of bacillus subtilis acetylglucosamine

A technology of Bacillus subtilis and recombinant bacteria, applied in the field of genetic engineering, can solve problems such as metabolic overflow and NADH consumption, and achieve the effects of simple construction method, reduced NADH, and easy use

Active Publication Date: 2018-08-21
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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  • Method for promoting synthesis of bacillus subtilis acetylglucosamine
  • Method for promoting synthesis of bacillus subtilis acetylglucosamine
  • Method for promoting synthesis of bacillus subtilis acetylglucosamine

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 synthesis of bacillus subtilis acetylglucosamine and belongs to the field of genetic engineering. The method is characterized in that a recombinant bacillus subtilis BSGNKAP2 is taken as a starting strain, and pyruvate carboxylase BalpycA from bacillus cereus is exogenously introduced, so that metabolism overflow of bacillus subtilis central carbon iseliminated and synthesis of acetoin as a byproduct is avoided; furthermore, five exogenous reductive metabolism reactions are introduced to replace a glycolytic pathway and a reaction of producing NADH in tricarboxylic acid cycle and reconstruct intracellular reductive metabolism, wherein the five exogenous reductive metabolism reactions specifically comprise glyceraldehydes-3-ferredoxin phosphate dehydrogenas, isocitric acid NAD<+> dehydrogenas, malic acid quinone dehydrogenas, ketonic acid ferredoxin oxidordeuctase and nitrogenase ferritin. In the shake-flask fermentation process of using acomplex medium, the yield of a recombinant strain BSGNKAP8 acetylglucosamine is 24.50g / L, and the yield of acetylglucosamine / glucose is 0.469g / L, which are 1.97 times and 2.13 times that of the starting strain BSGNKAP2.

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 Applications(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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