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High-yield acetyl-glucosamine recombinant bacillus subtilis and construction method thereof

A technology of Bacillus subtilis and acetamido, applied in the field of genetic engineering, can solve the problems of people who are not suitable for seafood allergy, the products are easy to cause allergic reactions, environmental pollution, etc., achieve good application prospects, strengthen the synthesis route, and are easy to use. Effect

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

At present, acetyl glucosamine 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.

Method used

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  • High-yield acetyl-glucosamine recombinant bacillus subtilis and construction method thereof
  • High-yield acetyl-glucosamine recombinant bacillus subtilis and construction method thereof
  • High-yield acetyl-glucosamine recombinant bacillus subtilis and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of Bacillus subtilis BSGN6-P xylA -glck

[0031]According to the sequence analysis of the glucokinase coding gene glck of Bacillus subtilis (Bacillus subtilis 168, purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, the glck promoter region was obtained, and the promoter replacement was designed according to its upstream and downstream sequences Frame homology arm amplification primers, left arm upstream and downstream primers are: glck-L-F: 5'-AGATTCCTGATGTTTTTCTTGCTCGAA-3'; glck-L-R: 5'-AGGATCCCCGGGTACCGAGCTCGACAAAAAAGCCAGCTTCCTTTCC-3';

[0032] The upper and lower primers of the right arm are: glck-R-F: 5'-CACTTAAATCAAAGGGGGAAATGTACAATGGACGAGATATGGTTTGCGG-3'; glck-R-R: 5'-TTAACAATTTTGATGTTTCAGCCATTC-3'.

[0033] The above primers were used to amplify the left and right arms contained in the replacement frame from the Bacillus subtilis genome. The specific conditions for the amplification were: pre-denaturation at...

Embodiment 2

[0037] Construction of recombinant Bacillus subtilis BSGN6-P xylA -glck -P xylA -pgi

[0038] According to the sequence analysis of the phosphoglucose isomerase coding gene pgi of Bacillus subtilis (Bacillus subtilis 168, purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, the pgi promoter region was obtained. According to its upstream and downstream sequences The primers for homologous arm amplification of the promoter replacement frame were designed. The upstream and downstream primers of the left arm were: pgi-L-F: 5'-ATTTGGAGGTTTTACTATGGAAAATTTCA-3'; pgi-L-R: 5'-AGGATCCCCGGGTACCGAGCTCTCCTTCAAGCGCAATTTCTTTATCT-3'; the upstream and downstream primers of the right arm were For: pgi-R-F:5'-CACTTAAATCAAAGGGGGAAATGTACAATGACGCATGTACGCTTTGACTACTC-3' and pgi-R-R:5'-GCTTCTCTACGTTCAGGACCGTTTC-3'. Use the above primers to amplify the left and right arms contained in the replacement frame from the Bacillus subtilis genome. The specific conditi...

Embodiment 3

[0042] Using the recombinant Bacillus subtilis constructed in Example 2 to ferment and produce acetylglucosamine

[0043] The components of the seed medium include: 10g / L peptone, 5g / L yeast powder and 10g / L sodium chloride.

[0044] 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 and 10ml / L trace element solution.

[0045] Wherein the trace element solution includes the following components based on its weight: 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.

[0046] In the seed medium, recombinant Bacillus subtilis BSGN6-P xylA -glck -P xylA -pgi was cultured at 37°C and 220rpm for 9 hours, and then the seeds were transferred to the fermentation medium ...

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Abstract

The invention relates to a high-yield acetyl-glucosamine recombinant bacillus subtilis and a construction method thereof. Bacillus subtilis BSGN6 serves as an original strain, glucokinase encoding gene glck expression and glucose phosphate isomerase encoding gene pgi expression in the bacillus subtilis are regulated by xylose inducible promoters PxylA, moderate expression of glck and pgi genes is controlled, the yield of acetyl-glucosamine in the recombinant bacillus subtilis is greatly improved, and a foundation is laid for further producing glucosamine by modifying the bacillus subtilis in metabolic engineering. The construction method is simple and easy to operate, the yield of acetyl-glucosamine can be greatly improved by the constructed recombinant bacillus subtilis, and the recombinant bacillus subtilis 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 with high acetylglucosamine production and a construction method thereof. 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 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 environmen...

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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