Graduation date: 2007Molecular genetic and enzymological techniques have been employed to study antibiotic biosynthesis. The nonproteinogenic amino acid capreomycidine is the signature residue found in the tuberactinomycin family of antitubercular peptide antibiotics and an important element of the pharmacophore. Recombinant VioG, a single module peptide synthetase from the viomycin gene cluster cloned from Streptomyces vinaceus (ATCC11861), is shown to specifically activate capreomycidine for incorporation into viomycin (tuberactinomycin B). Insertional gene disruption of the putative hydroxylase gene vioQ resulted in a mutant that accumulated tuberactinomycin O, confirming that hydroxylation at C-5 of the capreomycidine residue is a post-assembly event. The inactivated chromosomal copy of vioQ could be complemented with a wild-type copy of the gene to restore viomycin production. Chimeric genes have been constructed in an attempt to generate viomycin analogs containing enduracididine residue in place of capreomycidine. The expression of these genes resulted in insoluble proteins. Further investigation is needed to obtain functional chimeric NRPS modules to produce viomycin analogs containing enduracididine.