Microcin J25 (MccJ25) is an antimicrobial peptide produced by isolates of Escherichia colt with activity against closely related species. Production and export of mature MccJ25 in E. coli requires four genes that are organized on a plasmid-borne cluster in natural producer strains. In these strains, MccJ25 production does not commence until the cells reach stationary phase, and, according to previous literature, the highest titers of MccJ25 are obtained from cells grown in nutrient-poor medium. We sought to design an engineered MccJ25 gene cluster that alleviated the growth phase and media limitations of the natural cluster. In contrast to previous reports, we observe here that production of MccJ25 from its natural cluster is efficient in rich media, such as Luria Bertani (LB). The engineered gene cluster functions in several E. coli strains and produces titers of MccJ25 that are moderately increased (1.5- to 2-fold) relative to the natural cluster. RT-PCR experiments and translational GFP fusion experiments confirm that the engineered cluster produces MccJ25 throughout exponential phase. Furthermore, we provide evidence that control of the natural MccJ25 gene cluster is at the transcriptional level. The observations herein provide design parameters for large-scale production of MccJ25 for biotechnological applications. (C) 2009 Elsevier Inc. All rights reserved.