A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.