Modification and de novo design of non-ribosomal peptide synthetases (NRPS) using specific assembly points within condensation domains

Abstract

Many important natural products are produced by non-ribosomal peptide synthetases (NRPSs) 1.These giant enzyme machines activate amino acids in an assembly line fashion in which a set of catalytically active domains is responsible for the section, activation, covalent binding and connection of a specific amino acid to the growing peptide chain 1,2. Since NRPS are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would give access to a diverse range of peptides available biotechnologically. Here we describe a new fusion point inside condensation (C) domains of NRPSs that enables the efficient production of peptides, even containing non-natural amino acids, in yields higher than 280 mg/L. The technology called eXchange Unit 2.0 (XU2.0) also allows the generation of targeted peptide libraries and therefore might be suitable for the future identification of bioactive peptide derivatives for pharmaceutical and other applications.