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Promoter replacement method for improving Bacillus amyloliquefaciens yield

A technology of bacillus and starch liquefaction, applied in the biological field, can solve the problem of low production of antimicrobial peptides

Inactive Publication Date: 2009-07-08
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] However, the general antimicrobial peptide production of common bacterial strains is very low. So far, the production of lipopeptide producing bacteria is generally lower than 1.0g / L, and even less than 0.1g / L. Therefore, it is necessary to carry out mutagenesis to the production strains. There have been a few attempts in this regard, and good results have been achieved (Journal of Natural Products, 2002, 63: 1492-1496)

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  • Promoter replacement method for improving Bacillus amyloliquefaciens yield

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Embodiment Construction

[0043] The realization of the present invention is by cloning a homologous fragment (containing promoter Ppps) of a synthetase gene fenA of a synthetase gene fenA of fengycin of Bacillus amyloliquefaciens ES-2 genome DNA, and this fragment is passed SOE PCR method and start Sub P 59 Ligated, and the fragments connected together were cloned into the plasmid pMD19-T to obtain the plasmid pMD19-T1, and finally the neomycin resistance gene was connected to the EcoR I restriction site of the plasmid pMD19-T1 as a selection marker, thereby A homologous integration vector pMD19-T2 was constructed. And the homologous integration vector was transformed into Bacillus amyloliquefaciens ES-2, and a strain with strong promoter replacement was obtained. Detection of fengycin by the Oxford cup method and high performance liquid chromatography to identify the substitution strain with increased yield.

[0044] (1) Promoter P 59 sequence cloning

[0045] The constitutive promoter P was ampl...

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Abstract

The invention relates to a promoter replacement method for improving the output of the fecula liquefied bacillus fengycin, belonging to the field of biotechnology. The method includes: using a PCR method to amplify and obtain a 450bp homologous sequence from fecula liquefied bacillus ES-2 strain genome, amplifying a promoter P59 sequence from a plasmid pHB201, connecting together the two DNA fragments, and cloning into a plasmid pMD19-T, and connecting a neomycin resistance gene as a selection marker, thereby constructing a homologous integration vector; and then transforming the constructed homologous integration vector into the wild fecula liquified bacillus ES-2, wherein, the fengycin output of the promoter strain is 5704.77 +- 258.89mg / L, which improves about 0.65 times than the fengycin output of the wild bacteria.

Description

1. Technical field [0001] The invention relates to a promoter replacement method for improving the yield of fengycin of bacillus amyloliquefaciens, which is a method for improving the yield of lipopeptide antibiotic fengycin of bacillus amyloliquefaciens by utilizing the promoter replacement technology, and belongs to the field of biotechnology. 2. Background technology [0002] [Abstract] Since Arima (Biochemical and Biophysical Research Communication, 1968, 31: 488-496.) first discovered that Bacillus subtilis (B.subtilis) can produce antibacterial lipopeptide surfactin in 1968, the screening of antibacterial lipopeptide producing bacteria has mainly focused on Between different strains of Bacillus subtilis (Chinese Agricultural Sciences, 2003, (12): 1496-1501. Acta Microbiology, 2003, 43 (5): 647-652.). In recent years, further studies have shown that, in addition to Bacillus subtilis, Bacillus amyloliquefaciens (B.amyloliquefaciens) (Phytichemistry, 2002, 61:693-698.), B...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/11C12N1/21C12Q1/68C12Q1/04C12R1/07
Inventor 陆兆新孙会刚别小妹吕凤霞乌云达来卢亚萍王煜曹林
Owner NANJING AGRICULTURAL UNIVERSITY
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