Method for increasing yield of antifungal peptide bacillomycin D by overexpressing degU gene

An antifungal peptide, overexpression technology, applied in microorganism-based methods, other methods of inserting foreign genetic materials, genetic engineering, etc., can solve the problems of little bacillomycinD and high production costs

Active Publication Date: 2018-12-28
NANJING AGRICULTURAL UNIVERSITY +1
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Problems solved by technology

However, chemically synthesized preservatives have food safety risks. Research and development of new natural and safe agricultural product preservatives are of great significance in ensuring food and food safety and preventing fungal contamination.
But in practice, it has not been widely used in sustainable agriculture and environmental protection. The reason is that its production cost is high

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  • Method for increasing yield of antifungal peptide bacillomycin D by overexpressing degU gene
  • Method for increasing yield of antifungal peptide bacillomycin D by overexpressing degU gene
  • Method for increasing yield of antifungal peptide bacillomycin D by overexpressing degU gene

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specific Embodiment approach

[0033] The present invention first constructs the degU gene overexpression vector pCBS-LPDegUR, transforms the pCBS-LPDegUR vector into CGMCC No.0943 Bacillus subtilis (Bacillus subtilis) fmbJ through electrotransformation, and undergoes a double exchange process of homologous recombination in its genome. The degU gene with a promoter is inserted into the gene site to achieve the purpose of overexpressing the degU gene, thereby constructing a Bacillus subtilis strain fmbJdegU with high production of antimicrobial peptides. The mutant strains were identified by PCR method; the yield of bacillomycin D of the improved strain was analyzed by high performance liquid chromatography (HPLC). The specific implementation is as follows:

[0034] (1) Construction of degU gene overexpression vector pCBS-LPDegUR

[0035]Primers DegU-F and DegU-R were designed according to the Bacillus subtilis degU gene (Gene ID: 5459660), and the complete degU gene (702bp) with restriction sites KpnI and ...

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Abstract

The invention provides a method for increasing yield of antifungal peptide bacillomycin D by overexpressing a degU gene and belongs to the technical field of biology. The method comprises steps as follows: firstly, a gene overexpression vector pCBS-LPDegUR is constructed by bacillus subtilis fmbJ with preservation number being CGMCC No.0943 as an original strain and an escherichia coli-bacillus subtilis shuttle cloning vector pMAD as a skeleton, and by means of the homologous recombination principle, a strain fmbJdegU with high bacillomycin D yield is constructed with a method for knocking theDegU gene of bacillus subtilis fmbJ genome into an amylase site in a double exchange process. High performance liquid chromatography determines that capacity of the degU gene overexpressing strain for producing bacillomycin D is improved substantially.

Description

1. Technical field [0001] The invention relates to a method for increasing the output of antifungal peptide bacillomycin D by overexpressing degU gene, and belongs to the field of biotechnology. 2. Background technology [0002] As rhizosphere microbes that promote plant growth, Bacillus spp. show great promise in sustainable agriculture. They can not only secrete substances that promote plant growth, but also produce many secondary metabolites with antibacterial activity. [0003] CGMCC No.0943 Bacillus subtilis fmbJ is a bacillus that can secrete bacillomycinD, surfactin, fengycin and other antibacterial lipopeptide substances. Among them, bacillomycin D belongs to the iturin family member, and is a cyclic lipopeptide structure formed by condensation of a β-amino fatty acid with 14-17 C and 7 amino acids catalyzed by non-ribosomal polypeptide synthetase. It has a strong inhibitory effect on Aspergillus flavus, A. graminearum and Fusarium graminearum. Among them, mycotox...

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/66C12N15/90C12P21/00C12R1/125
CPCC07K14/32C12N15/66C12N15/75C12N15/902
Inventor 陆兆新孙静吕凤霞朱筱玉李金良
Owner NANJING AGRICULTURAL UNIVERSITY
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