Genomic library screening and genome mining are currently employed to identify biosynthetic gene clusters of thiopeptides. To elucidate the biosynthetic pathway of nocathiacin, we present a new approach with the application of simple reverse genetics. A relationship between structural features of thiopeptides and their biosynthetic pathways is established and is a starting point for speedily elucidating biosynthetic genes of various ribosomally-synthesized bioactive peptides with diverse modifications.