Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment

Kenji Tsuge, Kuniko Matsui, Mitsuhiro Itaya

Research output: Contribution to journalArticle

29 Citations (Scopus)


Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6 kb), and sfp (1.0 kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40 kb.

Original languageEnglish
Pages (from-to)592-603
Number of pages12
JournalJournal of Biotechnology
Issue number4
Publication statusPublished - 2007 May 10



  • Bacillus subtilis
  • Gene assembly
  • NRPS
  • OGAB
  • Plasmid

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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