Lantibiotics are antimicrobial peptides that contain several unusual amino acids resulting from a series of enzyme-mediated posttranslational modifications. As a consequence of being gene-encoded, the implementation of peptide bioengineering systems has the potential to yield lantibiotic variants with enhanced chemical and physical properties. Here we describe a functional two-plasmid expression system which has been developed to allow random mutagenesis of the two-component lantibiotic, lacticin 3147. One of these plasmids contains a randomly mutated version of the two structural genes, ltnA1 and ltnA2, and the associated promoter, Pbac, while the other encodes the remainder of the proteins required for the biosynthesis of, and immunity to, lacticin 3147. To test this system, a bank of similar to 1,500 mutant strains was generated and screened to identify mutations that have a detrimental impact on the bioactivity of lacticin 3147. This strategy established/confirmed the importance of specific residues in the structural peptides and their associated leaders and revealed that a number of alterations which mapped to the -10 or -35 regions of Pbac abolished promoter activity. Copyright (c) 2007 S. Karger AG, Basel.