Non-ribosomal peptide synthesis is a major source of natural, medically active compounds used to treat serious bacterial infectious, cancer and that function as immune suppressive agents. Peptide synthesis by non-ribosomal peptide synthetase (NRPS) assembly lines typically relies on array of large, repetitive units called modules, which themselves are comprised of various catalytic domains. Each module usually contains at least three core domains: these ensure selective activation of a specific, desired amino acid, transfer of the activated substrate onto a phosphopantetheine linker of a carrier protein and peptide bond formation between two adjacent activated substrates. Due to the modular architecture of NRPSs, there is great interest in understanding the biochemical basis of peptide synthesis by these machineries and further to enable their reprogramming to produce novel peptides with improved bioactivity. To date, however, such reengineering attempts have had limited success due to our incomplete knowledge of these complex systems.

In this study, we have concentrated on the reconstitution of the antibiotic teicoplanin NRPS assembly line reassembly in vitro and have commenced the analysis of critical, fundamental enzymatic steps involved in teicoplanin biosynthesis. Here, we present the results of our teicoplanin biosynthesis reconstitution system together with the discovery of a novel mechanism by which NRPS assembly lines ensure the correct modified state of specific of amino acids are incorporated into peptide chain even in the presence of competing substrates. The results of our study highlight new possibilities for the redesign of the glycopeptide antibiotics produced by NRPS assembly lines along with many other important compounds produced by these mega-synthase machineries.