Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes

Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.