Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu 12 ) and kynurenine (Kyn 13 ) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu 12 module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp 13 or Ile 13 at high efficiencies. A recombinant expressing DptD with a hybrid Kyn 13 module containing a di-domain from a D-Asn module caused the production of a new daptomycin analogue containing Asn 13 .

Daptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as CubicinH (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the DdptD mutation.