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Method for increasing antifungal peptide bacillomycin D yield by expressing comA gene

An antifungal peptide, overexpression technology, applied in microorganism-based methods, biochemical equipment and methods, peptides, etc., can solve the problems of little bacillomycinD and high production costs

Active Publication Date: 2019-01-04
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. Therefore, increasing its output has become a key issue to reduce its production cost.
[0004] At present, research results at home and abroad show that the method to increase the production of bacillomycin D is mainly to screen excellent strains and optimize their fermentation conditions, while the method of improving the production of bacillomycin D by means of molecular biology is rarely used

Method used

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  • Method for increasing antifungal peptide bacillomycin D yield by expressing comA gene
  • Method for increasing antifungal peptide bacillomycin D yield by expressing comA gene
  • Method for increasing antifungal peptide bacillomycin D yield by expressing comA gene

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specific Embodiment approach

[0033] The present invention first constructs the comA gene overexpression vector pCBS-LPComAR, and transforms the pCBS-LPComAR vector into Bacillus subtilis (Bacillus subtilis) fmbJ (commonly known and public, China Microbiological Culture Collection Management Committee General Microbiology Center, CGMCC No.0943) through electroporation ), through the homologous recombination double exchange process, the comA gene with a promoter was inserted into the amylase gene locus in its genome to achieve the purpose of overexpressing the comA gene, thereby constructing a Bacillus subtilis strain fmbJcomA with high production of antimicrobial peptides. The mutant strain was 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 comA gene overexpression vector pCBS-LPComAR

[0035]Primers ComA-F and ComA-R were designed according t...

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Abstract

The invention relates to a method for increasing antifungal peptide bacillomycin D yield by expressing comA gene and belongs to the technical field biology. The method is characterized in that CGMCC No. 0943 bacillus subtilis fmbJ is used as the original strain and escherichia coli-bacillus subtilis shuttle cloning vector pMAD is used as the framework to build a gene overexpression vector pCBS-LPComAR, and a high-bacillomycin-D-yield strain named fmbJcomA is built by knocking in the bacillus subtilis fmbJ genome comA gene at an amylase site during double exchange according to the homologous recombination principle. High-performance liquid chromatography determines that the bacillomycin D production capacity of the improved strain is increased greatly.

Description

1. Technical field [0001] The invention discloses a method for increasing the output of the antifungal peptide bacillomycin D by overexpressing the comA gene, in particular to a method for increasing the output of the antifungal peptide bacillomycin D of Bacillus subtilis by overexpressing the comA 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] Bacillus subtilis fmbJ is a bacillus that can secrete bacillomycin D, 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 po...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12P21/02C12R1/125
CPCC07K14/32C12N15/75C12P21/02
Inventor 陆兆新孙静别小妹吕凤霞赵海珍李金良
Owner NANJING AGRICULTURAL UNIVERSITY
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