Thiopeptides are structurally complex, bioactive natural products derived from ribosomally synthesized and post-translationally modified peptides. A remarkable set of enzymes were recently revealed to catalyze the formation of the core trithiazolylpyridine of thiopeptides via a formal [4 + 2] cycloaddition. These pyridine synthases typically act late in thiopeptide biosynthesis to affect macrocyclization and cleavage of the N-terminal leader peptide, making them potentially useful biocatalysts for preparation of new thiopeptide variants. Herein we investigate the leader peptide requirements for TcIM from thiocillin biosynthesis in Bacillus cereus ATCC 14579. Through a series of truncations, we define a minimum recognition sequence (RS) that is necessary and sufficient for TclM activity. This RS can be readily synthesized and ligated to linear thiopeptide cores prepared via solid-phase peptide synthesis (SPPS), giving an efficient and modular route to thiopeptide variants. We exploit this strategy to define C-terminal core peptide requirements and explore the differences in promiscuity of two pyridine synthases, TclM and TbtD, ultimately examining their ability to access new structural variants.