Promotor replacement method for improving volume of production of bacillus subtilis surfactin

A Bacillus subtilis and promoter technology, applied in the biological field, can solve the problem of low fermentation yield of surfactin in industrial production, and achieve the effect of increasing yield

Inactive Publication Date: 2009-04-08
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although the production level of surfactin can be improved through the optimization of fermentation process and mutation breeding, the fermentation yield of surfactin is still very low compared with industrial production

Method used

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  • Promotor replacement method for improving volume of production of bacillus subtilis surfactin

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Embodiment Construction

[0024] The present invention is realized by cloning a synthetase srfA of surfactin of Bacillus subtilis fmbR (Bie Xiaomei et al. Bacillus subtilis fmbR antibacterial substances. Chinese Agricultural Sciences, 2006, (11): 2327-2334) -A gene ( figure 1 Shown) 5' end has the homologous fragment (SEQ IDNO.1) of promoter Psrf, and size is 1081bp, and this fragment is connected on the pMUTIN4 vector with promoter, transforms bacillus subtilis fmbR then, and pass Erythromycin resistance screened the strains with promoter replacement. Finally, the yield of surfactin was detected by high performance liquid phase, so as to obtain the replacement strain with improved yield of surfactin.

[0025] (1) Vector pMUTIN4 containing promoter Pspac

[0026] Select the carrier pMUTIN4 (purchased from BGSC) with the IPTG-inducible promoter Pspac to construct a homologous integration vector. There is a multiple cloning site downstream of the promoter Pspac on the vector pMUTIN4, and a homologous f...

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Abstract

The invention relates to a promoter replacement method for improving the yield of Bacillus subtilis surfactin, belonging to the biotechnology field. The DNA homologous integration technology is adopted for allowing a promoter Pspac to replace the promoter of a Bacillus subtilis fmbR surfactin synthase gene. The homologous sequence of 1081bp is obtained by amplification from an fmbR strain genome through the PCR method and connected to HindIII and BamH I restriction enzyme cutting sites of a plasmid pMUTIN4, a well constructed vector is converted into wild Bacillus subtilis fmbR, thereby realizing the replacement through the screening by an antibiotic culture medium and the molecular biology method. The method can directly improve an antimicrobial peptide production strain on genetics, thereby having potential application value in industry. The yield of the strain surfactin is improved by about 4.85 times, and the yield of the surfactin can be improved by about 10 times under the IPTG induction.

Description

1. Technical field [0001] The invention relates to a promoter replacement method for improving the yield of bacillus subtilis surfactin, which is a method for improving the yield of lipopeptide antibiotic surfactin of bacillus subtilis by using the promoter replacement technology, and belongs to the field of biotechnology. 2. Background technology [0002] Bacillus subtilis can produce different kinds of antibacterial substances during growth and metabolism, among which antimicrobial peptides are dominant. [Abstract] The antibacterial peptides produced by Bacillus subtilis are usually divided into two categories: one is lantibiotics synthesized by ribosomes, mainly including subtilin, Erisin, sublancin, and subtilosin, etc. (Applied Microbiol Biotechnol, 1997, 48 : 80-82. Journal Biotenology, 2002, 5: 1-8. Archive Microbiology, 1996, 165: 243-251. J. Biol. Chem. 1998, 273: 23134-23142. Rev Microbiol, 1993, 47: 535-564 .), usually inhibit the growth of Gram-positive bacteria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12Q1/68C12Q1/04C12R1/125
Inventor 陆兆新孙会刚别小妹吕凤霞王煜曹林
Owner NANJING AGRICULTURAL UNIVERSITY
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