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Method for increasing yield of antifungal peptide bacillomycin D by over-expression of spo0A gene

An antifungal peptide, overexpression technology, applied in the direction of peptide, bacteria, recombinant DNA technology, etc., can solve the problems of little bacillomycinD and high production cost

Active Publication Date: 2018-12-18
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

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  • Method for increasing yield of antifungal peptide bacillomycin D by over-expression of spo0A gene
  • Method for increasing yield of antifungal peptide bacillomycin D by over-expression of spo0A gene
  • Method for increasing yield of antifungal peptide bacillomycin D by over-expression of spo0A gene

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specific Embodiment approach

[0033] The present invention first constructs the spo0A gene overexpression vector pCBS-LPSpo0AR, and transforms the pCBS-LPSpo0AR vector into Bacillus subtilis (Bacillus subtilis) fmbJ (commonly known and publicly used, Common Microbiology Center of China Microbial Species Collection Management Committee, CGMCC No. ), after the homologous recombination double exchange process, insert the spo0A gene with the promoter into the amylase gene site in the genome to achieve the purpose of overexpressing the spo0A gene, thereby constructing a Bacillus subtilis strain fmbJspo0A with high production of antimicrobial peptides. The mutant strain was identified by PCR; the yield of the modified strain bacillomycin D was analyzed by high performance liquid chromatography (HPLC). The specific implementation is as follows:

[0034] (1) Construction of spo0A gene overexpression vector pCBS-LPSpo0AR

[0035] The primers Spo0A-F and Spo0A-R were designed according to the Bacillus subtilis spo0A gen...

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Abstract

The invention relates to a method for increasing yield of antifungal peptide bacillomycin D by over-expression of spo0A gene and belongs to the field of biotechnology. In the method, firstly, with CGMCC No.0943 Bacillus subtilis fmbJ being an initial strain, a gene over-expression carrier pCBS-LPSpo0AR is constructed with Escherichia coli-Bacillus subtilis shuttle cloning carrier pMAD being a framework, and by means of the principle of homologous recombination, a strain, fmbJspo0A, which is high in yield of bacillomycin D, is constructed in a manner of knocking the spo0A gene of the fmbJ genome of the Bacillus subtilis in an amylase locus through a double exchange process. A high-performance liquid chromatographic test proves that the capability of producing the bacillomycin D by the improved bacterial strain is increased by 1.82 times.

Description

1. Technical Field [0001] The invention relates to a method for increasing the production of antifungal peptide bacillomycin D by overexpressing spo0A gene, and belongs to the field of biotechnology. 2. Background technology [0002] As rhizosphere microorganisms that promote plant growth, Bacillus has shown bright prospects 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 (Bacillus subtilis) fmbJ is a bacillus that can secrete antibacterial lipopeptides such as bacillomycin D, surfactin, and fengycin. Among them, bacillomycin D belongs to the iturin family and is a cyclic lipopeptide structure formed by the condensation of a 14-17 C β-amino fatty acid and 7 amino acids, catalyzed by non-ribosomal polypeptide synthetase. It has a strong inhibitory effect on Aspergillus flavus, Aspergillus ochraceus and Fusarium graminearum. Among them, my...

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N1/21C12P21/02C07K7/56
CPCC07K7/56C12N15/66C12N15/70C12P21/02
Inventor 陆兆新孙静吕凤霞别小妹张充李金良
Owner NANJING AGRICULTURAL UNIVERSITY
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