Microcins compose a class of antimicrobial peptides produced by enterobacteria, distinguished by their low molecular masses, to exert potent activity against closely related bacteria.  They are synthesized ribosomally, enabling direct genetic engineering to improve their activities.  A member of this class of peptides, microcin J25 (MccJ25), has an unusual lasso structure that confers exceptional resistance to high temperature, proteases, and chaotropic agents.  Its stability along with its compelling mechanism of action – inhibition of RNA polymerase – makes MccJ25 interesting for biotechnological and pharmaceutical applications.

Consistent with its ecological role, however, the production of MccJ25 takes place during stationary phase of growth, limiting the yield of active peptide to only 1-3 mg/L of culture medium.  The transcription of the structural gene, mcjA, is induced only at the onset of stationary phase as a result of nutrient depletion.  In contrast, the transcription of the genes required for maturation (mcjB and mcjC) and for export (mcjD) occurs during all stages of growth.  To improve the yield of MccJ25, we have re-engineered the gene cluster to create a novel expression system.  Preliminary data indicate that the engineered system offers improved yield and adaptability for antibiotic production compared to the natural system.  This high-yield, robust system for the production of MccJ25 also serves as a platform to further study the peptide to generate improved and novel functions.