Rational Design of Peptide Antibiotics by Targeted Replacement of Bacterial and Fungal Domains

Abstract

Peptide synthetases involved in the nonribosomal synthesis of peptide secondary metabolites possess a highly conserved domain structure. The arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product. A general approach has been developed for targeted substitution of amino acid-activating domains within the srfA operon, which encodes the protein templates for the synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. Exchange of domain-coding regions of bacterial and fungal origin led to the construction of hybrid genes that encoded peptide synthetases with altered amino acid specificities and the production of peptides with modified amino acid sequences.

Non-ribosomal peptide synthetase module fusions to produce derivatives of daptomycin in Streptomyces roseosporus

“…Module exchanges in NRPS model systems have proven to be useful to generate peptides of a targeted sequence (Doekel & Marahiel, 2000;Mootz et al, 2000), and several novel derivatives of surfactin have been produced by the targeted replacement of modules (Mootz et al, 2002;Stachelhaus et al, 1995;Yakimov et al, 2000). In other in vivo studies, the specificity-conferring codes in the active sites of A domains (Stachelhaus et al, 1999;Challis et al, 2000) have been exploited to redirect surfactin biosynthesis by site-specific mutagenesis (Eppelmann et al, 2002).…”

“…The linkers have been used in vitro (Doekel & Marahiel, 2000;Mootz et al, 2000) and in vivo (Stachelhaus et al, 1995;Nguyen et al, 2006b) to engineer modular NRPSs by module and domain exchanges. Production levels of novel lipopeptides related to daptomycin, engineered by single module exchanges, ranged from 17 to 50 % of controls ).…”

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: Sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates

“…It is unknown whether paired A-PCP domains communicate with each other largely because of covalently enforced proximity (the in cis fusion within a module), which ensures a high local concentration of Ppant-SH to capture the aminoacyl-AMP lodged in the adjacent A domain, or whether there is editing specificity for the particular aminoacyl moiety to be captured and͞or proteinprotein recognition features between a given A domain and its immediate downstream PCP domain. The general answer has implications both for the efficiency and design fidelity of any NRPS assembly line as well as for domain swap strategies in combinatorial biosynthesis approaches to create diversity in NRPS antibiotic peptides (8,9).To begin to dissect paired A-PCP domain recognition issues, we have turned to the Escherichia coli enterobactin synthetase system, an NRPS comprised of the three proteins EntE, EntB, and EntF (10, 11) (Fig. 2).…”

Editing of non-cognate aminoacyl adenylates by peptide synthetases

“…The distraction of the non-cognate aminoacyl adenylate was assayed as an excessive hydrolysis of ATP to AMP and PP i in the ATP pyrophosphatase reaction. These control steps may pose limitations for the application of such enzyme systems in the production of novel peptides by recombinational integration of alternative amino acid-activating modules [20].…”