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Recombinant bacillus subtitles capable of increasing yield of acetylglucosamine

A technology of Bacillus subtilis and acetamido is applied in the field of genetic engineering to achieve the effects of extracellular improvement, cell growth recovery, and yield and yield improvement

Inactive Publication Date: 2018-02-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Bacillus subtilis is a Gram-positive bacterium, which does not have the periplasmic space structure of Escherichia coli, so it is speculated that in the recombinant Bacillus subtilis BSGNKAP-PxylA-glmS-P43-GNA1, 6-phosphoacetylglucosamine is converted into acetylamino The reaction of glucose may be the rate-limiting step in its synthesis of acetylglucosamine

Method used

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  • Recombinant bacillus subtitles capable of increasing yield of acetylglucosamine
  • Recombinant bacillus subtitles capable of increasing yield of acetylglucosamine
  • Recombinant bacillus subtitles capable of increasing yield of acetylglucosamine

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of recombinant Bacillus subtilis BSGNY-P xylA -glmS -P 43 -GNA1

[0029] According to the upstream and downstream sequences of the nagAB gene of Bacillus subtilis (Bacillus subtilis 168 purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI and the phosphatase coding gene yqaB (nucleotide sequence such as NCBI-Gene ID: 945776 ), the terminator λT 0 , strong constitutive promoter P veg , and the sequence of the chloramphenicol resistance gene, construct the sequence as shown in SEQ ID NO.1 expression cassette, integrate the phosphatase yqaB into the nagAB site of the genome and use the strong constitutive promoter P veg to express.

[0030] Transform the constructed expression cassette into BSGNKAP-P xylA -glmS -P 43 -GNA1 (i.e. the recombinant Bacillus subtilis BSGNKAP constructed in patent application 201510761678.6), through chloramphenicol resistance plate screening and colony PCR verification, it was c...

Embodiment 2

[0031] Example 2: Construction of recombinant Bacillus subtilis BSGNY-P veg- glmS-P 43 -GNA1

[0032] According to the glmS gene and its upstream sequence of Bacillus subtilis (Bacillus subtilis 168 purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, the terminator λT 0 , strong constitutive promoter P veg , and the sequence of the chloramphenicol resistance gene, construct the glmS promoter replacement frame whose sequence is shown in SEQ ID NO.2, and replace the original promoter of glmS with the strong constitutive promoter P veg . Transform the constructed replacement frame into the recombinant Bacillus subtilis BSGNY-P in Example 1 xylA -glmS -P 43 -GNA1, through chloramphenicol resistance plate screening and colony PCR verification, it was confirmed that the recombinant Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 was obtained. Transform the plasmid pTSC (gifted by Dr. Yan Xin, Nanjing Agricultural University, NCBI accession n...

Embodiment 3

[0033] Example 3: Fermentation of recombinant Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 to produce acetylglucosamine

[0034] The recombinant bacillus constructed in Example 2 was used to verify the level of the 3L fermenter. The seeds cultivated at 37°C and 220rpm for 12h were transferred to the fermentation medium of the upper tank with a 5% inoculum size. Cultivate at 500-900 rpm for 52-72 hours. The content of acetylglucosamine in the final fermentation supernatant reaches 60.5g / L, and the yield of recombinant Bacillus subtilis 3L fermentation acetylglucosamine provided by the present invention is 0.311g / g glucose simultaneously, and the production intensity is 0.983g / L / h (results As shown in Table 1), the extracellular production of acetylglucosamine in recombinant Bacillus subtilis was improved, and the foundation was laid for further metabolic engineering of Bacillus subtilis to produce glucosamine.

[0035] Table 1 Cell growth and acetylglucosamine synthesis before a...

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Abstract

The invention discloses recombinant bacillus subtitles capable of increasing the yield of acetylglucosamine and belongs to the field of genetic engineering. The recombinant bacillus subtitles is characterized in that bacillus subtitles BSGNKAP-Pxy1A-glmS-P43-GNA1 is used as the original strain, a phosphatase yqaB gene controlled by a strong constitutive promoter Pveg is integrated to the genome, and the promoter of 6-phosphate glucosamine synthase is replaced by the strong constitutive promoter Pveg to obtain genetically engineered bacillus subtitles BSGNY-Pveg-glmS-P43-GNA1 accumulating the acetylglucosamine. The recombinant bacillus subtitles has the advantages that the yield of the acetylglucosamine of a 3L fermentation tank reaches 60.5g / L, the yield of the acetylglucosamine is 0.311g / g glucose, production intensity is 0.983g / L / h, the yield of the acetylglucosamine outside the cells of the recombinant bacillus subtitles is increased, and a foundation is laid for glucosamine production using metabolic engineering modified bacillus subtitles.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis with improved acetylglucosamine production, which belongs 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. 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 li...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/16C12P19/26C12R1/125
Inventor 刘龙陈坚堵国成李江华武耀康陈泰驰
Owner JIANGNAN UNIV
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