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Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine

A Bacillus subtilis, acetamido technology, applied in the field of genetic engineering, can solve the problems of low yield, low substrate/product molar conversion rate and the like

Active Publication Date: 2016-01-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

201510394205.7 Constructed recombinant Bacillus subtilis (BSGNK-P xylA -glmS -P 43 -GNA1) can grow faster in synthetic media and produce acetylglucosamine by fermentation, but has the disadvantages of low yield and low substrate / product molar conversion rate

Method used

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  • Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine
  • Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine
  • Recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Knockout of pyruvate kinase encoding gene pyK

[0026] According to the upstream and downstream sequences of the phosphoenolpyruvate carboxylase encoding gene pckA of Bacillus subtilis (Bacillus subtilis 168 purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, and the sequence of the bleomycin resistance gene , to construct a knockout box whose sequence is shown in SEQ ID NO.1.

[0027] Transform the constructed knockout frame into recombinant Bacillus subtilis BSGNK-P xylA -glmS -P 43-GNA1 (i.e. the recombinant Bacillus subtilis BSGNK constructed in patent application 201510394205.7), obtained recombinant Bacillus subtilis BSGNKA through bleomycin resistance plate screening and colony PCR verification; then further knocked out pyruvate on the basis of BSGNKA Kinase (pyk), the recombinant Bacillus subtilis BSGNKAP was obtained after successful knockout. Recombinant Bacillus subtilis BSGNK-P xylA -glmS -P 43 -GNA1 i...

Embodiment 2

[0028] Example 2: Fermentative production of acetylglucosamine

[0029] The seeds cultivated at 37° C. and 220 rpm for 12 hours were transferred to the fermentation medium at an inoculum size of 5%, and cultivated at 30-37° C. and 200-220 rpm for 36-52 hours. Fermentation 36h, acetylglucosamine content reaches 11.13g / L in the fermented supernatant liquid, compares with BSGNKA, and yield improves by 55.8%, compares with BSGNK, yield improves by 67.8% (results are shown in Table 1), realized Enhanced extracellular production of acetylglucosamine in recombinant Bacillus subtilis.

[0030] Table 1 Cell growth and acetylglucosamine synthesis before and after knockout

[0031]

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Abstract

The invention discloses recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine and belongs to the field of genetic engineering. According to the recombinant bacillus subtilis, based on bacillus subtilis BSGNK, the enol phosphate type pyruvate carboxylase encoding gene pckA and pyruvate kinase encoding gene pyk (NCBI upper Gene ID: 938206) are knocked out, the BSGNK regards B.subtilis168delta nagP delta gamP delta gamA delta nag A delta nag B delta ldh delta pta::lox <72> as the host, promoters PxylA and P43 are utilized for controlling recombinant ecpression of glmS and GNAl, and accordingly the glucose kinase encoding gene glcK is knocked out. According to the recombinant bacillus, glucose can be effectively utilized for synthesizing acetylglucosamine, the shake flask fermentation yield can reach 11.13 g / L, the yield is increased by 67.8% compared with a strain before knocking out, and a basis is provided for further improvement of bacillus subtilis for producing glucosamine of metabolic engineering.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis for efficiently synthesizing acetylglucosamine, 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 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, and the ...

Claims

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Application Information

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