Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus which is the active ingredient of CubicinŽ (daptomycin for injection). This first-in-class antibiotic was approved for the treatment of skin and skin-structure infections caused by Gram-positive pathogens in 2003 and bacteremia and endocarditis caused by Staphylococcus aureus in 2006. Daptomycin, however, is not an effective therapy for pulmonary pneumonia caused by Streptococcus pneumoniae. Daptomycin interacts with lung surfactant in vitro, and this interaction inhibits daptomycin's ability to kill bacteria. A54145 factors are a mixture of cyclic lipopeptides antibiotics produced by Streptomyces fradiae which, like daptomycin, are composed of 13 amino acids cyclized to form a 10-membered ring with a three exocyclic amino acids coupled to a lipid tail. The A54145 amino acid backbone differs significantly from daptomycin, but these lipopeptides also kill Gram-positive bacteria in a calcium dependent manner. A54145 factor D is not as potent as daptomycin in vitro, but shows activity in the presence of lung surfactant and is active in mouse pulmonary infection models. Using an integrative BAC vector system, we have manipulated the biosynthetic pathway genes required for the biosynthesis of the nonproteogenic amino acids, and used NRPS module exchanges to alter the peptide core. These novel A54145 lipopeptide analogs were evaluated for in vitro and in vivo activity versus target pathogens and these data will be discussed.