Polyketide (PK) and nonribosomal peptides (NRP), constructed on multimodular enzymatic assembly lines, often attain the conformations that establish biological activity by cyclization constraints introduced by tailoring enzymes. The dedicated tailoring enzymes are encoded by genes clustered with the assembly line genes for coordinated regulation. NRP heterocyclizations to thiazoles and oxazoles can occur on the elongating framework of acyl-S enzyme intermediates, whereas tandem cyclic PK polyether formation of furans and pyrans can be initiated by post-assembly line epoxidases. Macrocyclizations of NRP, PK, and hybrid NRP-PK scaffolds occur in assembly line chain termination steps. Post-assembly line cascades of enzymatic oxidations also create cross-linked and cyclized architectures that generate the mature scaffolds of natural product antibiotics. The modularity of the natural product assembly lines and permissivity of tailoring enzymes offer prospects for reprogramming to create novel antibiotics with optimized properties.