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).
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