Non-ribosomal peptide synthetases (NRPSs) are the origin of aw ide range of natural products,i ncluding many clinically used drugs.Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge.H ere we describe ac loning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L À1 .E xtending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains.T hese synthetic type of NRPS (type S) enables easier access to engineering,o vercomes cloning limitations,a nd provides as imple and rapid approach to building peptide libraries via the combination of different NRPS subunits.

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.

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.