The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) polyketide synthase (PKS) are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing, and macrocyclization. In these studies, the native pentaketide intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in engineered monomodules Ery5, Ery5-TE, and Ery6. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles and suggest that substrate recognition has evolved unique characteristics in each system.