Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof

A technology of Bacillus subtilis and glucosamine, which is applied in the field of genetic engineering, can solve the problems of environmental pollution, serious, unsuitable for people with seafood allergy, etc., and achieves the effects of easy use, simple construction method and good application prospect.

Inactive Publication Date: 2013-04-17
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. The waste liquid produced by this method is relativ

Method used

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  • Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof
  • Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof
  • Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof

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Experimental program
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Embodiment 1

[0022] The construction of embodiment 1 recombinant plasmid

[0023] According to the nucleotide sequence of the glucosamine acetylase encoding gene (GNA1) in Saccharomyces cerevisiae S288C (purchased from the American Type Microorganism Collection, No. ATCC 204508) published on NCBI, such as SEQ ID NO.1, the primer GNA1-F5' was designed -GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA1-R: 5'-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3'. The gene encoding glucosamine acetylase (GNA1) was amplified from the Saccharomyces cerevisiae S288C genome using the above primers. The amplified fragment was digested with KpnI and HindIII and then ligated into the pP43NMK expression vector (kindly provided by Dr. Zhang Xiaozhou, Virginia Tech, USA). Restriction digestion and sequencing confirmed the successful construction of the recombinant plasmid pP43-GNA1.

Embodiment 2

[0024] The construction of embodiment 2 recombinant Bacillus subtilis

[0025] The constructed expression vector pP43-GNA1 was transformed into Bacillus subtilis (Bacillus subtilis 168, purchased from Bacillus Genetic Collection, Ohio State University, USA). GNA1-F and GNA1-R primers were used to select transformants for colony PCR, and a 480bp band appeared, verifying that the recombinant Bacillus subtilis was successfully constructed.

Embodiment 3

[0026] Example 3 Fermentative production of acetylglucosamine

[0027] The seeds cultivated at 37°C and 200rpm for 12h were transferred to the fermentation medium with an inoculum size of 5%, and cultivated at 37°C and 200rpm for 30h. Finally, the content of acetylglucosamine in the fermentation supernatant reached 115mgL. The extracellular accumulation of acetylglucosamine in recombinant Bacillus subtilis was achieved by overexpressing the gene encoding glucosamine acetylase (GNA1).

[0028] Comparative Example 1 Integrated expression of GNA1 to accumulate acetylglucosamine

[0029] According to the pP43NMK plasmid sequence (GenBank accessionno.DQ264732) published on NCBI and the glucosamine acetylase coding gene (GNA1) in Saccharomyces cerevisiae S288C, the primer GNA1-2-F was designed: 5'-GGGGTACCTGATAGGTGGTATGTTTTCGCTTG-3', GNA1 -2-R: 5'-CGGGATCCCCTATTTTCTAATTTGCATTTCCACG-3'. The above primers were used to amplify the GNA1 expression cassette from pP43-GNA1, and the amp...

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Abstract

The invention discloses an acetyl-glucosamine accumulating recombinant bacillus subtilis and an application thereof, belonging to the field of genetic engineering. According to the invention, bacillus subtilis 168 is used as an expression host, a glucosamine acetylase coding gene (GNA1) derived from saccharomyces cerevisiae S288C is over-expressed, and the synthesis route of the glucosamine is strengthened so as to obtain the genetic engineering bacteria of the acetyl-glucosamine accumulating bacillus subtilis, wherein the yield is up to 115 mgL. The invention lays the foundation for the further metabolic engineering transformation of bacillus subtilis to produce the glucosamine.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis accumulating acetylglucosamine and an application thereof, belonging to the field of genetic engineering. 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 produced by acid-decomposing chitin in shrimp shells or crab shells. The waste liquid produced by this method is relatively serious for environmental pollution, a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/54C12P19/26C12R1/125
Inventor 陈坚堵国成刘龙李江华刘延峰
Owner JIANGNAN UNIV
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