One-pot synthesis of azoline-containing peptides in a cell-free translation system integrated with a posttranslational cyclodehydratase.

Azoline moieties in the backbones of peptidic natural products are important structural motifs that contribute to diverse bioactivities. Some azoline-containing peptides (Az-peptides) are produced from ribosomally synthesized precursor peptides, in which cysteine, serine, and threonine residues are converted to their corresponding azolines by posttranslational modification through a cyclodehydratase. We have devised an in vitro biosynthesis system of Az-peptides, referred to as the FIT-PatD (flexible in vitro translation) system, by the integration of a cell-free translation system with the posttranslational cyclodehydratase PatD. This system enabled the "one-pot" synthesis of a wide variety of Az-peptide derivatives expressed from synthetic DNA templates. The FIT-PatD system also facilitated mutagenesis studies on a wide array of precursor peptide sequences, unveiling unique in vitro substrate tolerance of PatD.