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Recombinant bacillus subtilis and method for promoting synthesis of acetylglucosamine by overexpressing glutamine synthetase by virtue of recombinant bacillus subtilis

A technology of Bacillus subtilis and glutamine, which is applied in the field of genetic engineering, can solve problems such as people who are not suitable for taking seafood allergies, products that easily cause allergic reactions, and environmental pollution, and achieve simple construction methods, improved transformation efficiency, and good application foreground effect

Inactive Publication Date: 2018-09-25
DAZIRAN BIOLOGICAL GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 the obtained products are easy to cause allergic reactions, so it is not suitable for people with seafood allergies.

Method used

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  • Recombinant bacillus subtilis and method for promoting synthesis of acetylglucosamine by overexpressing glutamine synthetase by virtue of recombinant bacillus subtilis

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Embodiment 1

[0024] The recombinant Bacillus subtilis, on the basis of the recombinant Bacillus subtilis BSGNK, further overexpresses the glutamine synthetase (EglnA) from Escherichia coli to obtain the recombinant BSGNKN; the recombinant Bacillus subtilis BSGNK is based on B. subtilis 168 Δ nagP Δ gamP Δ gamA Δ nagA Δ nagB Δ ldh Δ pta Δ glck :: lox72 as the host, respectively with the promoter P xylA ,P 43 control glms, GNA1 recombinant expression. The Escherichia coli glutamine synthetase (EglnA) coding gene glnA As shown in geneID: 948370 in NCBI, geneID: 948370 in NCBI is well known to those of ordinary skill in the art, and will not be described in detail here. The construction integration expression frame of the overexpression source Escherichia coli glutamine synthetase (EglnA) is SEQID NO.1, and is integrated into the glcK site through homologous recombination.

[0025] According to the Bacillus subtilis ( Bacillus subtilis 168 The upstream and downstream sequences of...

Embodiment 2

[0032] In this example, the recombinant bacterium BSGNKN is combined with fermentation medium to produce acetylglucosamine. Specifically, the seed culture solution of the activated recombinant Bacillus subtilis was transferred to the fermentation medium at an inoculation amount of at least 10% for inoculation, and an inducer was added after inoculation for at least 2 h and placed in a 500 mL shake flask within, at 37 o C for at least 48 h at 220 rpm. The inducer includes xylose, and the dosage of xylose is 5 g / L; the filling volume of the shake flask is 50 mL.

[0033] The fermentation medium of the present embodiment comprises the following components in g / L:

[0034] Initial glucose 60, peptone 6, yeast powder 12, (NH 4 ) SO 4 6. K 2 HPO 4 ·3H 2 O 12.5, KH 2 PO 4 2.5, CaCO 3 5. Trace element solution 10 ml / L; said trace element solution includes the following components in g / L: MnSO 4 ·5H 2 O1, Cocl 2 ·6H 2 O 0.4, NaMoO 4 2H 2 O 0.2, ZnSO 4 ·7H 2 O 0.2,...

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Abstract

The invention discloses recombinant bacillus subtilis. According to the recombinant bacillus subtilis, on the basis of the recombinant bacillus subtilis BSGNK, escherichia coli glutamine synthetase (EglnA) is overexpressed so as to obtain recombinant bacteria BSGNKN; and the recombinant bacillus subtilis BSGNK takes B. subtilis 168 delta nagP delta gamP delta gamA delta nagA delta nagB delta ldh delta pta delta glck::lox72 as a host, and the recombinant expression of glms and GNA1 is controlled by promoters PxylA and P43 respectively. The invention further discloses a method for promoting thesynthesis of acetylglucosamine by overexpressing the glutamine synthetase by virtue of the recombinant bacillus subtilis. The obtained recombinant bacillus subtilis has the advantages that the extracellular accumulation efficiency of the acetylglucosamine and the conversion efficiency of a substrate can be improved, the content of the acetylglucosamine in fermentation supernate is 7.8 g / L and is increased by 23.8% compared with the content of the acetylglucosamine of control bacteria BSGNK, a foundation is laid for further metabolic engineering modification of the bacillus subtilis for production of glucosamine, and in addition, the recombinant bacillus subtilis is simple in construction method and convenient to use and has a good application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant bacillus subtilis capable of increasing the yield of acetylglucosamine, and also relates to a method for using the recombinant bacillus subtilis to overexpress glutamine synthetase to promote the synthesis of acetylglucosamine. 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 produce...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/125
CPCC12N9/93C12P19/26C12Y603/01002
Inventor 徐恒德张西进朱玉钦刘家印李建波
Owner DAZIRAN BIOLOGICAL GRP CO LTD
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