In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues.

In nature, azolines produced by YcaO cyclodehydratases during the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) are generally limited to thiazoline, oxazoline, and methyloxazoline, which are derived from the proteinogenic Cys, Ser, and Thr residues, respectively. To investigate whether YcaO cyclodehydratases precisely recognize the common structural characteristics and chirality of the modifiable residues, the "reprogrammed FIT-PatD system" has been established by combining a YcaO cyclodehydratase (PatD) with genetic code reprogramming powered by the flexible in vitro translation (FIT) system, in which precursor peptides bearing non-proteinogenic Cys/Ser/Thr analogues could be expressed through a reprogrammed genetic code and subsequently cyclodehydrated by PatD. The study has revealed remarkable stereo-, chemo-, and regioversatility for modifiable residues in PatD-catalyzed cyclodehydration, expanding the repertoire of backbone heterocycles in RiPPs to exotic azoline analogues.