Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis

A technology of Bacillus subtilis and acetyl glucosamine, applied in the field of genetic engineering, can solve the problem of low acetyl glucosamine production, achieve the effects of reducing by-product formation, good application prospects, and improving accumulation

Active Publication Date: 2016-01-13
JIANGSU HEVI BIOTECH CO LTD
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  • 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 disadvantage of low acetylglucosamine yield (3g / L)

Method used

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  • Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis
  • Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis
  • Method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis

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Experimental program
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Embodiment 1

[0024] Example 1: Knockout of the gene pckA encoding phosphoenolpyruvate carboxylase

[0025] 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.

[0026] Transform the constructed knockout frame into recombinant Bacillus subtilis BSGNK-P xylA -glmS -P 43 -GNA1 (that is, the recombinant Bacillus subtilis BSGNK constructed in patent application 201510394205.7), through bleomycin resistance plate screening and colony PCR verification, confirmed that the knockout of phosphoenolpyruvate carboxylase (pckA) was successful, The recombinant Bacillus subtilis BSGNKA was obtained. Recombinant Bacillus subtilis BSGNK-P xylA -glmS -P43 -GNA1 is BSGN6-P constructed in the liter...

Embodiment 2

[0027] Example 2: Fermentative production of acetylglucosamine

[0028] The seeds cultivated at 37° C. and 220 rpm for 8 hours were transferred to the fermentation medium at an inoculum size of 3%, and cultured in a 3L fermenter at 35-37° C. and 600-800 rpm for 52-72 hours. When fermented for 52 hours, the content of acetylglucosamine in the fermentation supernatant reached 29.27g / L, which was different from that of the control bacterium BSGNK-P xylA -glmS -P 43 - Compared with GNA1, it increased by 28.1% (results are shown in Table 1), realizing the increase of the extracellular production of acetylglucosamine in recombinant Bacillus subtilis. In addition, the specific synthesis rate of acetylglucosamine of the BSGNKA bacterial strain of the present invention reaches 0.085g / gDCW / h, which is 46.5% higher than that of the control bacterial strain BSGNK. Simultaneously, the output of the by-product acetoin of the bacterial strain of the present invention is 23.5g / L, which is 6...

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Abstract

The invention discloses a method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis and belongs to the field of genetic engineering. The method comprises the steps that BSGNK-PxylA-glmS-P43-GNA1 serves as original strains, phosphoenolpyruvate carboxylase (pckA) genes are knocked out through homologous recombination, the reaction that enolphosphopyruvate is converted into oxaloacetic acid in host bacterium cells is blocked, the carbon flux flowing to a tricarboxylic acid cycle is reduced, and accumulation of acetylglucosamine is promoted. In the process that a composite culture medium is used for fermentation, the acetylglucosamine yield of recombination bacillus subtilis with pckA knocked out reaches 29.27 g/L and is increased by 28.1% compared with that of control strains. The method lays a foundation for transforming bacillus subtilis to produce acetylglucosamine in metabolic engineering.

Description

technical field [0001] The invention relates to a method for knocking out pckA to promote the synthesis of acetylglucosamine by Bacillus subtilis, 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 poll...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/26C12R1/125
Inventor 刘龙顾洋邓洁莹陈坚堵国成李江华
Owner JIANGSU HEVI BIOTECH CO LTD
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