The continued propagation of antibiotic resistance requires the development of new therapeutics. The lipopeptide antibiotic enduracidin has demonstrated high activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. In addition to a lack of cross-resistance with existing antibiotic classes, enduracidin has no known transferrable resistance mechanism. The development of enduracidin as a human therapeutic is hampered by its poor solubility in plasma. Utilizing chemical and genetic techniques, analogs of enduracidin have been produced and evaluated for biological activity. Making use of the hydroxyphenylglycine (Hpg) biosynthetic pathway, fluorine was incorporated into enduracidin with minimal to no loss of bioactivity. The semisynthetic chemical modification of enduracidin proved to be challenging. The chemical nitration of the Hpg residues was unsuccessful. Modifications to the lipid tail by cleavage at the C2-olefin with ozone and the use of Diels-Alder reagents to react with the lipid tail diene also proved unsuccessful. However, the reduction and dihydroxylation modifications of the lipid tail diene were successful. Introduction of polar hydroxyl groups onto the alkyl tail reduced bioactivity while reduction of the diene had no significant effect. Analysis of the biosynthetic pathways involved in producing the lipid tail and the unusual amino acid enduracididine yielded some insights into the formation of the antibiotic. Through complementation of mutants having disruptions in the biosynthetic gene cluster and crystallographic data, the function of EndR as a cyclase was established. Additionally, the use of 4-hydroxyarginine as an intermediate in enduracididine biosynthesis was demonstrated. The ability of EndQ to function as a transaminase on both 4-hydroxyarginine and 2-ketoenduracididine was also established. The specific functions of EndP and EndQ have not been determined. The introduction of the lipid tail diene by the three enzymes Orf39, Orf44 and Orf45 was confirmed. Orf45 functions as a CoA ligase and a dehydrogenase to introduce the C2 double bond. The functions of Orf39 and Orf44 appear to be the introduction of the C4 double bond and isomerization of the C2 olefin. / Graduation date: 2013