Cloning of srfA operon from Bacillus subtilis C9 and its expression in E. coli.
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 R(f) value of 0.52 from B. subtilis C9 or authentic surfactin (R(f) = 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.
Lee
Young-Ki
YK
Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, South Korea.
Yoon
Byung-Dae
BD
Yoon
Jung-Hoon
JH
Lee
Seung-Goo
SG
Song
Jae Jun
JJ
Kim
Jong-Guk
JG
Oh
Hee-Mock
HM
Kim
Hee-Sik
HS
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D055666:Lipopeptides; D009876:Operon; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D010456:Peptides, Cyclic / Q000378:metabolism; D010957:Plasmids / Q000235:genetics
D001426:Bacterial Proteins; D055666:Lipopeptides; D010456:Peptides, Cyclic; C009365:surfactin peptide; D010453:Peptide Synthases; C069542:surfactin synthetase