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Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same

A Bacillus subtilis and construction method technology, applied in the field of recombinant plasmid pDL-PSQ, can solve the problems of inability to produce Surfactin and Plipastatin, etc., and achieve the effect of high safety

Inactive Publication Date: 2016-05-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genome of B.subtilispB2 contains complete Surfactin synthase srfA operon and Plipastatin synthase pps operon, but due to base mutation inactivation of sfp gene and degQ gene, B.subtilispB2 cannot produce Surfactin and Plipastatin

Method used

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  • Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same
  • Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same
  • Surfactin and Plipastatin co-production bacillus subtilis strain and method for constructing same

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Embodiment 1

[0045] 1. Materials and methods

[0046] 1.1 Strains and plasmids

[0047] Bacillus subtilis B.subtilisfmbJ (CGMCCNO.0943), Escherichia coli Escherichiacoli DH5α cloning host (purchased from Novizan Biological Company), B.subtilispB2 and Bacillus subtilis integration plasmid pDL (YuanandWong, RegulationofgroEexpressioninBacillussubtilis:theinvolvementofthesigmaA-likepromoterandtherolesofttheinvertedrep9seinvertedrep9) ) were preserved by the Enzyme Engineering Laboratory of Nanjing Agricultural University; EscherichiacoliT-A cloning vector pMD19-T (purchased from TaKaRa Company). Indicator strains: peach soft rot pathogen Rhizopus stolonifer (AS3.2336), Micrococcus luteus (CMCC28001), purchased from the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences.

[0048] 1.2 Medium

[0049] LB medium: peptone 10.0g, yeast extract 5.0g, NaCl 10.0g, distilled water 1000mL, pH7.0~7.2;

[0050] 10×Tbaase: (NH 4 ) 2 SO 4 20.0g, K 2 HPO 4 140.0g,...

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Abstract

The invention belongs to the field of molecular biology and microbial fermentation, and provides a Surfactin and Plipastatin co-production bacillus subtilis strain pB2-L, a method for constructing the same and application of the Surfactin and Plipastatin co-production bacillus subtilis strain. The method for constructing the Surfactin and Plipastatin co-production bacillus subtilis strain includes steps of amplifying target genes; amplifying fusion fragments; acquiring intermediate vectors; acquiring recombinant integration plasmids; acquiring and detecting transformants. The Surfactin and Plipastatin co-production bacillus subtilis strain, the method and the application have the advantages that a fragment of fusion genes P43-sfp-degQ are led into amyE site of B-subtilis pB2 of non-producing antibacterial substances Surfactin and Plipastatin by the aid of homologous recombination processes, so that the recombinant Surfactin and Plipastatin co-production bacillus subtilis strain B.subtilis pB2-L can be obtained, the strain has certain antibacterial activity for gram positive bacteria and filamentous fungi, and the Surfactin and Plipastatin co-production bacillus subtilis strain, the method and the application have an important significance on follow-up study.

Description

technical field [0001] The invention belongs to the field of molecular biology and microbial fermentation, relates to a Bacillus subtilis strain co-producing Surfactin and Plipastatin, in particular to the Bacillus subtilis and its recombinant plasmid pDL-PSQ obtained in the construction process, the Bacillus subtilis Construction methods and their applications. Background technique [0002] Bacillus is a kind of Gram-positive bacteria widely distributed in nature. Among them, Bacillus subtilis and Bacillus amyloliquefaciens are dominant bacteria in soil and plant microecology, which can produce a variety of lipids that are widely used in plant disease control. Peptide antibacterial substances such as: Surfactin is a cyclic lipopeptide composed of a β-hydroxy fatty acid chain containing 13-14 carbon atoms and 7 amino acid peptides. As a strong biosurfactant, it also has Biological activities such as antibacterial, antitumor, antiviral and preventing fibrin clots. Fengycin ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75A01P1/00A01P3/00C12R1/125
CPCC07K14/32C12N9/1241C12N15/75C12N2800/101C12Y207/07003
Inventor 别小妹高玲陆兆新吕凤霞赵海珍张充
Owner NANJING AGRICULTURAL UNIVERSITY
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