Non-ribosomal peptide synthesis is a major source of medically active compounds - in particular compounds with antibiotic activity. In this process, peptide assembly is performed by large, modular enzymatic assembly lines known as non-ribosomal peptide synthetases (NRPSs). Great interest exists in understanding the biochemical and structural basis for peptide synthesis by NRPS machineries. This due to the fact that many current antibiotics are either natural products produced by NRPS machineries in bacteria, or are modified forms of these natural compounds. Interest is particularly high in exploring the possibility of reengineering these molecular assembly lines to enable the production of novel antibiotics. However, to date such reengineering attempts have had limited success due to incomplete knowledge of these complex systems.1
In this study, using amino acid activation assays2 and assembly line reassembly, we have reconstituted the activity of all NRPS modules from the biosynthesis of the antibiotic teicoplanin and demonstrated formation of the complete teicoplanin heptapeptide precursor biosynthesis in vitro. Importantly, we discovered that the 5th peptide bond forming-domain, which is responsible for formation of the 5th peptide bond in the linear heptapeptide precursor, is selective for modified amino acid substrates that rely on the interaction of external modification enzymes with the NRPS bound amino acid.
Our approach opens the door to more detailed NRPS reengineering and mechanistic analyses. Moreover, this reconstitution platform concept will provide important insights into the effect of alternate redesign strategies, such as domain swapping, module swapping, changes in amino acid activation domain selectivity etc., on different NRPS domains and build up a list of rules for effective NRPS redesign to enable the production of new bioactive compounds.