Recombinant bacillus subtilis and construction method and application thereof

一种枯草芽孢杆菌、氨基葡萄糖的技术，应用在遗传工程领域，能够解决加重细胞代谢负担、生产过程不稳定等问题，达到降低细胞代谢负担、构建方法简单、消除抑制的效果

Active Publication Date: 2018-12-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, the present invention uses the functional membrane microdomain of Bacillus subtilis cells itself as a space scaffold, utilizes the scaffold proteins FloA and FloT positioned in the functional membrane microdomain membrane raft, and anchors the enzyme on the space scaffold, solving the problem of existing protein scaffolds. Increase the burden of cell metabolism and cause instability in the production process. Compared with recombinant bacteria whose enzymes are not anchored on the space scaffold, the fermentation yield of acetylglucosamine is greatly improved

Method used

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  • Recombinant bacillus subtilis and construction method and application thereof
  • Recombinant bacillus subtilis and construction method and application thereof
  • Recombinant bacillus subtilis and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Verify that the FMMs of Bacillus subtilis can be used as a stable space scaffold

[0034]On the basis of Bacillus subtilis BSGN6, a fusion gene expression frame of FloT and green fluorescent protein EGFP was integrated at the site of the scaffold protein coding gene floT (NCBI-Gene ID: 937138) of Bacillus subtilis BSGN6 genome. Using the integration site floT sequence (length 1kb, remove the C-terminal stop codon), EGFP gene sequence (0.7kb) (nucleotide sequence shown in SEQ ID NO.2), bleomycin resistance gene zeo sequence ( The nucleotide sequence is shown in SEQ ID NO.3), FloT downstream sequence (1 kb in length) to construct the integration frame. Through homologous recombination, the integration frame obtained above was integrated into the genome of Bacillus subtilis BSGN6. Through bleomycin resistance plate screening, colony PCR verification, and sequencing, it was confirmed that the integration was successful and the recombinant Bacillus subtilis expre...

Embodiment 2

[0036] Example 2: Verification that Bacillus subtilis scaffolding proteins FloA and FloT are located in the same FMMs

[0037] On the basis of Bacillus subtilis BSGN6, the fusion gene expression cassette of FloA and green fluorescent protein EGFP was integrated at the site of the scaffold protein coding gene floA (NCBI-Gene ID: 937865) of Bacillus subtilis BSGN6 genome. Use the integration site floA sequence (length 1kb, remove the C-terminal stop codon), EGFP gene sequence (0.7kb), chloramphenicol resistance gene CmR sequence (nucleotide sequence shown in SEQ ID NO.4), FloA The downstream sequence (length 1 kb) constructs the integration frame. Through homologous recombination, the integration frame obtained above was integrated into the genome of Bacillus subtilis BSGN6. Through chloramphenicol resistance plate screening, colony PCR verification, and sequencing, it was confirmed that the integration was successful and the recombinant Bacillus subtilis expressing FloA-EGFP w...

Embodiment 3

[0040] Embodiment 3: Construction of recombinant Bacillus subtilis BSGAT

[0041] On the basis of Bacillus subtilis BSGN6, FloT and GlcN-6-P synthetase coding gene glmS (nucleotide sequence such as NCBI -Gene ID: shown in 938736) fusion gene expression cassette. Use the integration site floT sequence (length 1kb, remove the C-terminal stop codon), the glmS gene sequence (1.8kb) with (GGGGS)3linker at the N-terminus, the chloramphenicol resistance gene CmR sequence, and the downstream sequence of FloT (length 1kb ) to construct the FloT-GlmS fusion gene integration frame. Through homologous recombination, the integration frame obtained above was integrated into the genome of Bacillus subtilis BSGN6. Through chloramphenicol resistance plate screening, colony PCR verification, and sequencing, it was confirmed that the integration was successful to obtain recombinant Bacillus subtilis expressing FloT-GlmS, and the chloramphenicol resistance was knocked out.

[0042] On the basi...

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Abstract

The invention discloses a recombinant bacillus subtilis and a construction method and an application thereof, which belong to the technical field of genetic engineering. The method takes cell self functional membrane microdomain FMMs as a space frame, and constructs a multienzyme complex by using the specific marker proteins FloA and FloT, an artificial substrate channel is constructed, the metabolic burden of the cells can be effectively reduced, and the multienzyme complex is attached to plasmalemma , which facilitates the transport of a product from the intracellular to the extracellular. The recombinant bacillus subtilis constructed by the method efficiently synthesizes GlcNAc without affecting cell life activities, and a toxic intermediate metabolite GlcN-6-P can also be confined to the vicinity of the plasmalemma to reduce or eliminate the inhibition of cell viability. During a shake flask fermentation of a composite medium, the yield of acetylglucosamine in contrast bacterial strain BSGC is only 0.45 g / L, and the BSGAT acetylglucosamine output is increased to 5.29g / L, which is 11.76 times that of the contrast bacterial strain. The recombinant bacillus subtilis construction method of the invention is simple and convenient to use, and has the good application prospect.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis and its construction method and application, belonging to the technical field of genetic engineering. 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 diet 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 for people with seafood allergies. [0003] Bac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/26C12R1/125
CPCC12N9/16C12N9/80C12N15/75C12P19/26C07K14/32C07K2319/00C12N1/20C12N9/1029C12N9/1096C12Y203/01004C12Y206/01016C12N15/62C12R2001/125C12N1/205
Inventor 刘龙吕雪芹堵国成李江华陈坚
Owner JIANGNAN UNIV
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