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A kind of genetically engineered bacteria with high lipopeptide production and its application

A technology of genetically engineering bacteria and lipopeptides, applied in the field of genetic engineering, to achieve the effect of increasing the production of lipopeptides

Active Publication Date: 2021-12-31
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Knocking out which spore synthesis gene to construct a spore-free engineered bacterium to increase the production of surfactin and reduce the potential safety hazards of Bacillus subtilis has not been reported

Method used

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  • A kind of genetically engineered bacteria with high lipopeptide production and its application
  • A kind of genetically engineered bacteria with high lipopeptide production and its application
  • A kind of genetically engineered bacteria with high lipopeptide production and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 is used to knock out the construction of the linear fragment left-bleo-right of the spore gene

[0079] Build process such as figure 1 As shown, the spoIVB gene and the bleomycin resistance gene are taken as examples.

[0080] According to the p7z6 plasmid sequence (NCBI: EU541492.1) published on the database NCBI, the bleomycin resistance gene fragment bleo, 650bp, with lox recognition sites at both ends was synthesized.

[0081] The Bacillus subtilis THY-7 genome was extracted using the Bacterial Genome Extraction Kit from Omega. Using the obtained genome as a template, PCR amplification was performed using the upstream primer left-F and the downstream primer left-R to obtain the left fragment of the homology arm located upstream of the spoIVB gene in the genome. With the THY-7 genome as a template, use upstream primer right-F and downstream primer right-R to carry out PCR amplification to obtain the homology arm right fragment located in the downstream of t...

Embodiment 2

[0130] Example 2 Construction of spoIVB-deficient genetically engineered bacteria

[0131] The left-bleo-right fragment used for knocking out the spoIVB gene constructed in Example 1 was transformed into competent cells of Bacillus subtilis THY-7 and THY-7 / Pg3-srfA by electroporation to obtain spoIVB-deficient cells Genetically engineered bacteria THY-7 / spoIVB:bleo, THY-7 / Pg3-srfA / spoIVB:bleo. Wherein, the preparation and electrotransformation of Bacillus subtilis THY-7 / Pg3-srfA competent cells adopt the method in Chinese patent document CN105400784A.

[0132] After thawing, take 100uL of the bacterial liquid and spread it on the LB solid medium containing 10-30μg / mL bleomycin, and place it upside down in a 37°C incubator for overnight culture, pick a single colony, and use the upstream and downstream primers left-F and right -R for PCR verification. At the same time, the genome of the original strain was used as a template and PCR with the same primers as a control. The orig...

Embodiment 3

[0137] Example 3 Production of lipopeptide surfactant—surfactin using genetically engineered bacteria B.subtilis THY-7 / Pg3-srfAΔspo0A

[0138] The genetically engineered bacteria THY-7 / Pg3-srfAΔspoOA of the spore-deficient type obtained in Example 2 was inoculated in LB liquid medium, and cultivated for 16h at 37°C and 200rpm to obtain the genetically engineered bacteria liquid;

[0139] Insert 5% into the shake flask equipped with 100mL fermentation medium, add 1mM IPTG when cultivating for 2-6h under the conditions of 37°C and 200rpm, continue to cultivate until 2d, and the fermentation ends.

[0140] The composition of the fermentation medium used is: sugar 30-100g / L, inorganic nitrogen source 10-50g / L, organic nitrogen source 0.5-3g / L, KH 2 PO 4 0.1-1g / L, Na 2 HPO 4 12H 2 O 0.5-0.3g / L, CaCl 2 0.002-0.01g / L, MnSO 4 ·H 2 O 0.002-0.01g / L, FeSO 4 ·7H 2 O 0.002-0.01g / L, pH 6.5-7.5.

[0141] Microscopic observation of cell spore morphology during the fermentation pro...

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Abstract

The invention discloses a genetically engineered bacterium with high lipopeptide production and its application, belonging to the technical field of genetic engineering. The genetically engineered bacteria with high lipopeptide production is constructed by inactivating at least one gene in the fourth and fifth stages of spore synthesis in the original strain. The present invention blocks the synthesis of spores by knocking out the genes related to the fourth and fifth stages of spore synthesis in the genome of lipopeptide-producing strains, thereby obtaining a genetically engineered bacterium without spores and significantly increasing the lipopeptide content. The genetically engineered bacteria obtained in the present invention can maintain a complete cell shape in a high-concentration surfactin environment; compared with the starting strain, no spores are produced during the fermentation process and the lipopeptide production is significantly improved, and the highest surfactin production in the shake flask reaches 9.9% g / L, which is 25% higher than the starting strain, and the cell activity is also enhanced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium with high lipopeptide production and its application. Background technique [0002] Lipopeptide biosurfactants are a class of amphoteric substances linked by hydrophilic cyclic oligopeptides and hydrophobic fatty acid chains with lactone bonds, and are mainly synthesized by microorganisms such as Bacillus and Streptomyces. Bacillus lipopeptides can be divided into surfactin, phenmusin, iturin, etc. Surfactin, which forms a unique "saddle" conformation at the air / liquid interface, has excellent surface activity, biodegradability and antibacterial activity. It has broad application prospects in the fields of oil exploration, biological control, medicine and daily chemicals. However, the low yield of surfactin produced by Bacillus fermentation limits the industrial production and application of surfactin. At the same time, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P21/00C12R1/125C12R1/085C12R1/38
CPCC12P21/00C07K14/21C07K14/32
Inventor 于慧敏王苗苗
Owner TSINGHUA UNIV
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