Cloning of srfA operon from Bacillus subtilis C9 and its expression in E. coli

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Cloning of srfA operon from Bacillus subtilis C9 and its expression in E. coli
Young Ki Lee; Byung Dae Yoon; Jung-Hoon Yoon; Seung Goo LeeJae Jun Song; J G Kim; Hee-Mock OhHee-Sik Kim
Bibliographic Citation
Applied Microbiology and Biotechnology, vol. 75, no. 3, pp. 567-572
Publication Year
The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different Rf value of 0.52 from B. subtilis C9 or authentic surfactin (Rf=0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.
Bacillus subtilisLA-PCRsrfA operonSurfactin
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Synthetic Biology and Bioengineering Research Institute > 1. Journal Articles
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > Cell Factory Research Center > 1. Journal Articles
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