Engineered biosynthesis of tetracycline analogs is an attractive option to accelerate the development of the next generation of tetracycline compounds exhibiting novel antibiotic and anticancer properties, as well as to overcome the current modes of antibiotic resistance. Tetracyclines are aromatic polyketides that are biosynthesized by bacterial type II polyketide synthases (PKS). We sequenced the entire gene cluster of oxytetracycline (oxy) PKS from Streptomyces rimosus, and revealed a total of twenty-one genes that are putatively involved in oxytetracycline biosynthesis. One of the distinguishing features of tetracyclines is the presence of an amide unit at one terminus of the polyketide backbone. In vivo reconstitution using Streptomyces coelicolor (CH999) revealed that the asparagine synthase homolog OxyD is necessary and sufficient for the biosynthesis and incorporation of the malonamate starter unit. An amidated polyketide (WJ35) was synthesized as the major product when the oxy minimal PKS, the C9 ketoreductase (OxyJ) and OxyD are coexpressed in CH999. We are also reconstituting the biosynthesis of pretetramid of oxytetracycline heterologous hosts by expressing the minimal oxy PKS, the initiation module and the immediate tailoring enzymes. Pretetramid is an important compound because it will confirm the role of the numerous cyclases in the oxy cluster and it can be subjected to modification by different tailoring enzymes. More novel tetracycline intermediates have been characterized. Important new biochemical insights of aromatic PKSs based on their structural analysis will be presented.