incorporated and activates the carboxylate with ATP to make the aminoacyl-AMP. Next, the A domain installs an aminoacyl group on the thiolate of the adjacent T domain. The condensation (C) domain catalyzes the peptide bond forming reaction, which elicits chain elongation. It joins an upstream peptidyl-S-T to the downstream aminoacyl-S-T (Figure 7). Chain elongation by one aminoacyl residue and chain translocation to the next T domain occurs in concert. The order of these domains is C-A-T. In some instances, an epimerization domain is necessary in those modules where L-amino acid monomers are to be incorporated and epimerized to D-amino acids. The domain organization in such modules is C-A-T-E. The first module has a three-domain C-A-T organization; these often occur in assembly lines that make N-acylated peptides. The first C domain catalyzes N-acylation of the initiating amino acid (tryptophan) while it is installed on T. An adenylating enzyme (Ad) catalyzes the condensation of decanoic acid and the N-terminal tryptophan, which incorporates decanoic acid into the growing peptide (Figure 3). The genes responsible for this coupling event are dptE and dptF, which are located upstream of dptA, the first gene of the Daptomycin NRPS biosynthetic gene cluster. Once the coupling of decanoic acid to the N-terminal tryptophan residue occurs, the condensation of amino acids begins, catalyzed by the NRPS. The first five modules of the NRPS are encoded by the dptA gene and catalyze the condensation of L-tryptophan, D-asparagine, L-aspartate, L-threonine, and glycine, respectively (Figure 4). Modules 6–11, which catalyze the condensation of L-ornithine, L-aspartate, D-alanine, L-aspartate, glycine, and D-serine are encoded for the dptBC gene (Figure 5). dptD catalyzes the incorporation of two nonproteinogenic amino acids, L-3-methylglutamic acid (mGlu) and Kyn, which is only known thus far to daptomycin, into the growing peptide (Figure 6). Elongation by these NRPS modules ultimately leads to macrocyclization and release in which an α-amino group, namely threonine, acts as an internal nucleophile during cyclization to yield the 10-amino-acid ring (Figure 6). The termination module in the NRPS assembly line has a C-A-T-TE organization. The thioesterase domain catalyzes chain termination and release of the mature lipopeptide. The molecular engineering of daptomycin, the only marketed acidic lipopeptide antibiotic to date (Figure 8), has seen many advances since its inception into clinical medicine in 2003. It is an attractive target for combinatorial biosynthesis for many reasons: second generation derivatives are currently in the clinic for development; Streptomyces roseosporus,