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Bacillus subtilis genetic engineering bacterium for producing lipopeptide and construction method and application thereof

A technology of Bacillus subtilis and genetically engineered bacteria, applied in the field of biological genetic engineering, can solve problems such as low yield of antibacterial lipopeptide, and achieve the effects of improving production efficiency and reducing production costs

Inactive Publication Date: 2019-01-25
JIANGSU LOPALTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the yield of antimicrobial lipopeptides produced by fermentation of natural strains is low

Method used

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  • Bacillus subtilis genetic engineering bacterium for producing lipopeptide and construction method and application thereof
  • Bacillus subtilis genetic engineering bacterium for producing lipopeptide and construction method and application thereof
  • Bacillus subtilis genetic engineering bacterium for producing lipopeptide and construction method and application thereof

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Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of genetically engineered Bacillus subtilis LPB-4 containing recombinant plasmid pHT-srfA

[0042] Using the Bacillus subtilis genome as a template, primers were designed to clone the srfA gene. After correct analysis, it was connected with the E. coli-Bacillus subtilis shuttle plasmid pHT-trxA using Gebson assmbly technology. For the present invention, its concrete steps are as follows:

[0043] (1) Obtaining of srfA gene sequence and construction of recombinant plasmid:

[0044] Pick a single colony of Bacillus subtilis (the strain has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on December 4, 2017, the preservation number is CGMCC No.14375, and the strain number is LPB-3), and inoculated in LB liquid In the culture medium, culture at a temperature of 37°C and a shaker speed of 200rpm for 16h, centrifuge at 12000rpm for 5min to collect the bacterial pellet, use the bacter...

Embodiment 2

[0060] Embodiment 2: The lipopeptide biosurfactant produced by the genetically engineered Bacillus subtilis

[0061] Seed medium composition (g / L): glucose 15, ammonium sulfate 5, magnesium sulfate 2, potassium chloride 1, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 1.

[0062] Fermentation medium composition (g / L): glucose 30, ammonium sulfate 10, magnesium sulfate 2, potassium chloride 1, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 1, glycine 0.2, vitamin B12 0.1.

[0063] A ring of Bacillus subtilis LPB-4 strain was inoculated into LB test tube slant medium for activation, the culture temperature was 32°C, and the culture was statically cultured for 24 hours. The activated slant strain was transferred into a 2L shake flask for primary seed expansion cultivation at a cultivation temperature of 32° C., a shaking table rotation speed of 150 rpm, and a cultivation period of 10 h. According to the inoculum amount of 1% (volume perce...

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Abstract

The invention discloses a Bacillus subtilis genetic engineering bacterium for producing lipopeptide and a construction method and application thereof. Bacillus subtilis lipopeptide surfactant is use to synthesize key operon srfA gene and Escherichia coli-Bacillus subtilis shuttle plasmid so as to construct Bacillus subtilis genetic engineering strain which can produce lipopeptide, and it was applied in the fermentation of Bacillus subtilis engineering strain to produce lipopeptide surfactant, so as to obtain high concentration of lipopeptide surfactant. The highest yield of lipopeptide surfactant was 3.78 g / L, which was 47.7% higher than that of the original strain.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a lipopeptide-producing Bacillus subtilis genetically engineered bacterium and a construction method and application thereof. Background technique [0002] Bacillus subtilis is a Gram-positive bacterium used in the production of green biological products. It can secrete a variety of low-molecular-weight antimicrobial peptides and biosurfactants with potential biomedical and industrial applications, such as through non-ribose Low-molecular-weight antimicrobial lipopeptide surfactant (surfactin), iturin, fengycin, etc. synthesized through the ribosome pathway; bacteriocin (TasA) synthesized through the ribosome pathway. In recent years, with the wide application of genetic engineering technology, the biosynthetic mechanism of B. subtilis antibacterial active substances has also been deeply studied. Among all the antibacterial substances secreted by B. subtilis, antimi...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12P21/00C12R1/125
CPCC12N15/75C12P21/00C07K14/32
Inventor 史莹飞文昌卡尔·汉斯乔伊·马克斯
Owner JIANGSU LOPALTECH