BIOSYNTHESIS AND GENETIC ENGINEERING OF LIPOPEPTIDES IN STREPTOMYCES ROSEOSPORUS

Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics. Daptomycin biosynthesis is carried out by a giant multisubunit, multienzyme nonribosomal peptide synthetase (NRPS). The daptomycin (dpt) biosynthetic genes have been cloned in a bacterial artificial chromosome (BAC) vector, sequenced, and expressed in Streptomyces lividans. Several of the dpt genes, including the three NRPS genes, are transcribed as a lengthy polycistronic message. The daptomycin-producing strain, Streptomyces roseosporus, can be genetically manipulated, and a number of deletion mutants encompassing one or more of the dpt genes have been constructed. Several of the dpt genes have been expressed from ectopic chromosomal loci (phi C31 or IS117 attB sites) under the transcriptional control of the strong constitutive ermEp* promoter, and recombinant strains produced high levels of lipopeptides, thus establishing a trans-complementation system for combinatorial biosynthesis. A number of hybrid NRPS subunits have been generated by gamma-Red-mediated recombination, and combinatorial libraries of lipopeptides have been generated by NRPS subunit exchanges, module exchanges, multidomain exchanges, deletion mutagenesis, and multiple natural lipidations, using the ectopic trans-complementation system in S. roseosporus.