A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.