Unveiling the Biosynthetic Logic of Nosiheptide Based on Reconstitution of Its Bicyclic Thiopeptide Scaffold

Thiopeptides, which share a macrocyclic framework characterized by a six-membered, nitrogen heterocycle central to multiple (thi)azol(in)es and dehydroamino acids, represent one of the most structurally complex groups of ribosomally synthesized and post-translationally modified peptides (RiPPs). Although post-translational modifications (PTMs) necessary for common framework formation were established, how bicyclic thiopeptides, which depend on additional specific enzyme activities to afford a side ring system, are formed remains poorly understood. Using the biosynthesis of nosiheptide as a model system, here, we report the first PTM logic to achieve a bicyclic thiopeptide based on in vivo and in vitro structural reconstitution. Eleven biosynthetic proteins are employed, processing the precursor peptide through the proper coordination of five PTM steps, of which three are common and two are specific: (1) formation of five thiazoles, (2) incorporation of an indolic moiety, (3) dehydration of five Ser/Thr residues, (4) indolic side ring closure, and (5) pyridine formation to establish the thiopeptide framework. Heterologous expression and biochemical characterization validated that the two macrocyclic ring systems are established in an interdependent and alternating manner. Distinct from tailoring PTMs, this study unveils a paradigm of a new PTM introduction for expanding the chemical and biological spaces during the establishment of the group-defining framework.