6-deoxyerythronolide B (6-dEB) is the polyketide precursor to the broad-spectrum antibiotic erythromycin. Production has been metabolically engineered through Escherichia coli and optimized through media and process parameter variation. However, little work has been done to more thoroughly examine biosynthesis on the cellular level. Here, we report the incorporation of proteomic and metabolomic tools to better understand heterologous 6-dEB production with the goal of better understanding and maximizing production at the cellular level.