Lasso-type peptides exhibit a complex structure consisting of a side chain-to-backbone cyclization forming an amide bond. The C-terminal tail is entrapped within the ring, where it is sterically blocked by bulky side chains from residues on each side of the ring. The resulting lasso-type structure confers a particular stability to the peptides. Most of these naturally-occurring peptides were isolated from Streptomyces species and shown to be enzyme inhibitors. Microcin J25 (MccJ25) is such a lasso-type gene-encoded antibacterial peptide secreted by E. coli AY25. It uses the iron-siderophore receptor FhuA to enter bacteria and then inhibits RNA-polymerase in the cytoplasm. The mcjABCD gene cluster required for MccJ25 biosynthesis has been entirely sequenced: mcjA encodes the linear precursor of MccJ25 and mcjD is required for both MccJ25 export and self-immunity towards MccJ25. The last two genes mcjB and mcjC encode proteins that are putatively involved in the posttranslational modification and three-dimensional structure acquisition of the microcin. Using MccJ25, we have investigated here the biosynthesis of lasso peptides. Requirement for both mcjB and mcjC to obtain mature MccJ25 was shown by gene inactivation and complementation assays. McjA (precursor of MccJ25), McjB and McjC (putative modification enzymes) were subsequently obtained by expression in recombinant E. coli. The biosynthesis of MccJ25 was followed by both liquid chromatography coupled to mass spectrometry (LC-MS) and antibacterial assays. We showed that two enzymes only, McjB and McjC, are sufficient to achieve the conversion of the precursor McjA into mature MccJ25. To our knowledge, this is the first example of the in vitro reconstitution of a lasso-type peptide biosynthesis.