Recombinant bacillus subtilis capable of improving yield of acetylglucosamine, and constructing method thereof

A technology of Bacillus subtilis and acetamido, applied in the field of genetic engineering, can solve problems such as low conversion efficiency of acetylglucosamine/glucose, and achieve the effects of simple construction method, improved yield and good application prospect

Active Publication Date: 2016-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The recombinant Bacillus subtilis BSGNKAP constructed in the patent application No. 201510761678.6 still has the disadvantage of low conversion efficiency of acetylglucosamine / glucose

Method used

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  • Recombinant bacillus subtilis capable of improving yield of acetylglucosamine, and constructing method thereof
  • Recombinant bacillus subtilis capable of improving yield of acetylglucosamine, and constructing method thereof
  • Recombinant bacillus subtilis capable of improving yield of acetylglucosamine, and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of Bacillus subtilis BSGNKAP

[0034] According to the content disclosed in Chinese patent application 201510761678.6, B. subtilis 168Δ nagPΔ gamPΔ gamAΔ nagAΔ nagBΔ ldhΔ pta::lox72 was used as the host, and the promoter P xylA ,P 43 Control the recombinant expression of glmS and GNA1, free express GNA1 gene with pP43-GNA1 plasmid, integrate and express glmS gene with pM7Z6M-PxylA-glmS plasmid, and then knock out the glucokinase coding gene glck and phosphoenolpyruvate carboxylation on this basis Enzyme coding gene pckA and pyruvate kinase coding gene pyk are used to obtain recombinant Bacillus subtilis BSGNKAP.

Embodiment 2

[0036] Construction of recombinant Bacillus subtilis BSGNKAP2

[0037] According to the upstream sequence and pycA sequence of the pyruvate carboxylase coding gene pycA of Bacillus subtilis 168 purchased from the American Type Microorganism Collection Center with the number ATCC No.27370 published on NCBI (as shown in the sequence table SEQ ID NO.1) , PH01 terminator Ter, P 43A strong constitutive promoter and the sequence of the bleomycin resistance gene zeo are constructed to construct a replacement frame whose sequence is shown in SEQ ID NO.2. In the present invention, the expression regulation of the original promoter of pycA is terminated by introducing the terminator Ter of the PHT01 plasmid.

[0038] The constructed replacement frame was transformed into the Bacillus subtilis BSGNKAP obtained in Example 1, and after homologous recombination, the constructed replacement frame was integrated into the Bacillus subtilis BSGNKAP genome to replace the initiation of the pyruv...

Embodiment 3

[0040] Production of Acetyl Glucosamine by Fermentation of Recombinant Bacillus BSGNKAP2

[0041] The components of the seed medium include: 10g / L peptone, 5g / L yeast powder, and 10g / L sodium chloride.

[0042] The components of the fermentation medium include: 20g / L glucose, 6g / L peptone, 12g / L yeast powder, 6g / L ammonium sulfate, 12.5g / L dipotassium hydrogen phosphate, 2.5g / L potassium dihydrogen phosphate, 5g / L Calcium carbonate 10ml / L trace element solution.

[0043] The trace element solution is based on its weight, including the following components: 1.0g / L manganese sulfate, 0.4g / L cobalt chloride, 0.2g / L sodium molybdate, 0.2g / L zinc sulfate, 0.1g / L chloride Aluminum, 0.1g / L copper chloride, 0.05g / L boric acid and 5mol / L hydrochloric acid.

[0044] Use high performance liquid chromatography to detect the content of acetylglucosamine, high performance liquid chromatography test conditions: instrument model Agilent 1200, RID detector, column: NH 2 Column (250×4.6 mm, ...

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Abstract

The invention relates to a recombinant bacillus subtilis capable of improving the yield of acetylglucosamine. The recombinant bacillus subtilis is obtained by strengthening the expression of pyruvic carboxylase encoding gene pycA of the acetylglucosamine. The invention also provides a constructing method for the recombinant bacillus subtilis. The constructing method comprises the following steps: constructing a replacing frame of a promoter and an initiation codon of the pyruvic carboxylase encoding gene pycA; replacing an original promoter of the pyruvic carboxylase encoding gene pycA in a bacillus subtilis genome by a P43 strong constitutive promoter in the replacing frame through homologous recombination; meanwhile, replacing an initiation codon GTG of the pycA with ATG. Compared with a starting strain, the yield of the acetylglucosamine of the recombinant bacillus subtilis provided by the invention is greatly improved; the constructing method for the recombinant bacillus subtilis is simple and has an industrial value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant bacillus subtilis for improving the production of acetylglucosamine and a construction method thereof. 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 wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12P19/26C12R1/125
Inventor 刘龙顾洋邓洁莹陈坚堵国成李江华
Owner JIANGNAN UNIV
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