A mutagenesis study of the downstream recognition sequence (dRS) in the substrate peptide of a post-translational cyclodehydratase, PatD, was conducted by means of an in vitro reconstituted translation system integrated with PatD, named the FIT-PatD system. The results have revealed that PatD tolerates various dRS sequences, enabling the one-pot biosynthesis of azoline-containing peptides (Az-peptides) with various C-terminal tag sequences such as hexahistidine, FLAG epitope, and Avi-tag.