This study confirmed the participation of a cryptic palmitoyl fatty acyl chain in the biosynthesis of safracin and unraveled a previously ignored peptidase for the removal of the precursor. Furthermore, the post-assembly line tailoring steps are extensively studied in terms of the methyltransferase SacI-catalyzed N-methylation and the FAD-dependent monooxygenase SacJ-catalyzed A-ring oxidation. The timing of these post-NRPS steps is also addressed in this work.