Recombinant bacillus subtilis for improving yield of acetylglucosamine and building method of recombinant bacillus subtilis

A Bacillus subtilis, acetamido technology, applied in the field of genetic engineering, can solve the problems of low conversion efficiency of acetylglucosamine/glucose, insufficient supply of α-ketoglutarate, low carbon source utilization efficiency, etc., and achieves the promotion of acetylglucosamine. The effect of accumulation, reduction of by-product formation, and yield reduction

Active Publication Date: 2016-11-23
JIANGNAN UNIV
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Bacillus subtilis BSGNKAP constructed in the Chinese patent application No. 201510761678.6 still has the disadvantage of low conversion efficiency of acetylglucosamine / glucose
After BSGNKAP knocked out the gene encoding pyruvate kinase pyk and the gene encoding phosphoenolpyruvate carboxylase pckA, the intracellular malate repletion pathway of Bacillus subtilis will promote the conversion of malate into pyruvate, and pyruvate acts as a multiple The metabolic node of the metabolic pathway, so the pyruvate generated by malic acid replenishment will be further converted into acetoin, which will lead to excessive accumulation of by-products, resulting in low carbon source utilization efficiency
At the same time, due to the conversion of malate to pyruvate through the anaplerotic pathway, the supply of α-ketoglutarate, the precursor of acetylsamine synthesis, will also be insufficient.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant bacillus subtilis for improving yield of acetylglucosamine and building method of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for improving yield of acetylglucosamine and building method of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for improving yield of acetylglucosamine and building method of recombinant bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of Bacillus subtilis BSGNKAP

[0037]According to the content disclosed in Chinese patent application 201510761678.6, B. subtilis168ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔpta::lox72 was used as the host, and the promoter P xylA ,P 43 Controlling the recombinant expression of glmS and GNA1, free expression of GNA1 gene with pP43-GNA1 plasmid, pM7Z6M-P xylA The -glmS plasmid integrates and expresses the glmS gene, and then knocks out the glucokinase encoding gene glck, the phosphoenolpyruvate carboxylase encoding gene pckA and the pyruvate kinase encoding gene pyk on this basis, and obtains Bacillus subtilis BSGNKAP.

Embodiment 2

[0039] Construction of recombinant Bacillus subtilis BSGNKAPM1

[0040] According to the upstream and downstream sequences of the malate dehydrogenase gene ytsJ of Bacillus subtilis 168 (as shown in the sequence table SEQ ID NO.1) purchased from the American Type Microorganism Collection Center with the number ATCC No.27370 published on the NCBI, and Bo As for the sequence of the lyomycin resistance gene zeo, a gene knockout frame encoding malate dehydrogenase whose sequence is shown in SEQ ID NO.2 is constructed.

[0041] The constructed malate dehydrogenase encoding gene knockout frame was transformed into the Bacillus subtilis BSGNKAP obtained in Example 1, and the bleomycin resistance in the malate dehydrogenase encoding gene knockout frame was transformed through homologous recombination. The gene zeo replaced the malate dehydrogenase gene ytsJ in Bacillus subtilis BSGNKAP, which blocked the conversion of malate into pyruvate in the host bacteria. Through bleomycin resist...

Embodiment 3

[0043] Construction of recombinant Bacillus subtilis BSGNKAPM2

[0044] According to the upstream and downstream sequences of the malate dehydrogenase gene ywkA of Bacillus subtilis 168 (as shown in the sequence table SEQ ID NO.1) purchased from the American Type Microorganism Collection Center with the number ATCC No.27370 published on NCBI, and Bo As for the sequence of the lyomycin resistance gene zeo, a gene knockout frame encoding malate dehydrogenase whose sequence is shown in SEQ ID NO.2 is constructed.

[0045] The constructed malate dehydrogenase encoding gene knockout frame was transformed into the Bacillus subtilis BSGNKAPM1 obtained in Example 2, and the bleomycin resistance in the malate dehydrogenase encoding gene knockout frame was transformed through homologous recombination The gene zeo replaced the malate dehydrogenase gene ywkA in Bacillus subtilis BSGNKAPM1, which blocked the conversion of malate into pyruvate in the host bacteria. Through bleomycin resista...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides recombinant bacillus subtilis for improving the yield of acetylglucosamine. Malic dehydrogenase coding genes are knocked out from bacillus subtilis. The invention further provides a building method of the recombinant bacillus subtilis. The method comprises the steps that a malic dehydrogenase coding gene knock-out frame is built; malic dehydrogenase coding genes ytsJ, ywkA, malS and mleA are knocked out from a bacillus subtilis genome, and the recombinant bacillus subtilis for improving the yield of the acetylglucosamine is obtained. Compared with original strains, according to the recombinant bacillus subtilis, the acetylglucosamine extracellular accumulation amount is improved, and the yield of the by-product acetoin is reduced. The building method of the recombinant bacillus subtilis is simple, convenient to use and good in application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant bacillus subtilis for improving the production of acetylglucosamine and a construction method thereof. Background technique [0002] 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 dietary supplements 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 environmental pollution, and the obtained products are likely to cause allergic reactions, so it is not suitable...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap