Combinatorial biosynthesis of novel antibiotics related to daptomycin

Abstract

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. lambda-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.

Fig. 1.

Lipopeptide antibiotics and daptomycin gene cluster. (A) Structurally related lipopeptides daptomycin, A54145 and CDA. (B) Subunit and module organizations of the daptomycin gene cluster.

Fig. 2.

λ-Red-mediated recombination for construction of hybrid dptBC. The coding region of domains (CAT, in this example) of module d-Ala₈ was replaced by an antibiotic resistance cassette flanked by rare restriction sites. The cassette was then excised, and the linearized plasmid was used in ligation, at the compatible restriction sites, with the gap repair cloned fragments coding for other domains.

Fig. 3.

Daptomycin gene content in deletion mutants and expression plasmids. (A) NRPS deletion mutants as expression hosts. (B) Expression plasmids for hybrid NRPS pathways. Relative locations of domain and module exchanges on dptBC are marked by filled squares. Promoters that may drive expression of dpt subunit genes are indicated as bent arrows.

Fig. 4.

Amino acid sequence alignment of linkers of modules from the biosynthetic pathways of S. roseosporus daptomycin (Sr), S. fradiae A54145 (Sf), and Streptomyces coelicolor CDA (Sc). These modules incorporate the amino acid residues specified in single-letter codes on the left. Fusion sites are indicated. The * marks the stop of LptB followed by the first amino acid residue of LptC.