Two proteins, encoded by the tcmJ and tcmN genes, have been previously implicated in early cyclization steps in the biosynthesis of the aromatic polyketide tetracenomycin. In order to elucidate the function of these enzymes and to evaluate their potential for generating novel polyketides, several tcmJ- and tcmN-containing recombinant polyketide synthase (PKS) gene clusters were constructed and analyzed in vivo. These constructs led to the expression of combinations of the TcmJ and TcmN proteins with subunits from the actinorhodin (act) and tetracenomycin (tcm) PKSs, responsible for the biosynthesis of different polyketide backbones. In addition to three novel polyketides, RM77 (3), RM80 (4), and RM806b (5), which are characterized here, several previously isolated polyketides were produced. While results for TcmJ were inconclusive, comparison of the new molecules to other polyketide structures has allowed us to propose two functions for TcmN. First, consistent with earlier predictions [McDaniel, R.; Ebert-Khosla, S.; Fu, H.; Hopwood, D. A.; Khosla, C. Proc. Natl. Acad. Sci; U.S.A. 1994, 91, 11542-11546], the TcmN protein influences the regiospecificity of the intramolecular aldol condensation that forms the first ring. Second, the protein appears to catalyze the aromatization of the second ring, and is deduced to be a second ring aromatase. The inability of TcmN to have any affect on C-9 reduced backbones suggests that ketoreduction occurs prior to cyclization of the first ring in reduced polyketides. The analysis of TcmN extends the use of genetically engineered PKSs for novel polyketide design and biosynthesis.