Bacterial natural products in general, and non‐ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatized as inefficient, time‐, labour‐ and cost‐intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programs. As a follow‐up to our previously published proof‐of‐principle study on generating bipartite type S non‐ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non‐ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPSs in up to three subunits and rapidly generated different bi‐ and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mg L−1. A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re‐using previously created type S NRPS, but that functions of individual domains as well as domain‐domain interactions can be studied and assigned rapidly.

Many clinically used natural products are produced by non-ribosomal peptide synthetases (NRPSs), which due to their modular nature should be accessible to modification and engineering approaches. While the adenylation domain (A) plays the key role in substrate recognition and activation, the condensation domain (C) which is responsible for substrate linkage and stereochemical filtering recently became the subject of debate - with its attributed role as a "gatekeeper" being called into question. Since we have thoroughly investigated different combinations of C-A didomains in a series of in vitro, in vivo, and in situ experiments suggesting an important role to the C-A interface for the activity and specificity of the downstream A domain and not the C domain as such, we would like to contribute to this discussion. The role of the C-A interface, termed 'extended gatekeeping', due to structural features of the C domains, can also be transferred to other NRPSs by engineering, was finally investigated and characterised in an in silico approach on 30 wild-type and recombinant C-A interfaces. With these data, we not only would like to offer a new perspective on the specificity of C domains, but also to revise our previously established NRPS engineering and construction rules.

Bacterial natural products in general, and non-ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatised as inefficient, time-, labour-, and cost-intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new-to-nature natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programms. As a follow-up to our previously published proof-of-principle study on generating bipartite type S non-ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non-ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPS in up to three subunits and rapidly generated different bi- and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mgL-1. A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re-using previously created type S NRPS, but that functions of individual domains as well as domain-domain interactions can be studied and assigned rapidly.