The three-dimensional structure of natural products is critical for their biological activities and, as such, enzymes have evolved that specifically generate active stereoisomers. Lanthipeptides are post-translationally modified peptidic natural products that contain macrocyclic thioethers featuring lanthionine (Lan) and/or methyllanthionine (MeLan) residues with defined stereochemistry. In this report, we compare two class I lanthipeptide biosynthetic gene clusters (BGCs), coi and olv, that represent two families of lanthipeptide gene clusters found in Actinobacteria. The precursor peptides and BGCs are quite similar with genes encoding a dehydratase, cyclase, and methyltransferase (MT). We illustrate that the precursor peptide CoiA1 is converted by these enzymes into a polymacrocyclic product, mCoiA1, that contains an analogous ring pattern to the previously characterized post-translationally modified OlvA peptide (mOlvA). However, a clear distinction between the two BGCs is an additional Thr-glutamyl lyase (GL) domain that is fused to the MT, CoiSA, which results in divergence of the product stereochemistry for the coi BGC. Two out of three MeLan rings of mCoiA1 contain different stereochemistry than the corresponding residues in mOlvA, with the most notable difference being a rare d-allo-l-MeLan residue, the formation of which is guided by CoiSA. This study illustrates how nature utilizes a distinct GL to control natural product stereochemistry in lanthipeptide biosynthesis.