The mechanism of a type II polyketide synthase was analyzed by combinatorially expressing components of the tetracenomycin (tcm) and the actinorhodin (act) polyketide synthase genes in various mutants or heterologous hosts. Structural analysis of metabolites produced by the recombinant organisms provided evidence to dissect the function of individual components of a type II PKS. Complementation studies with the S. glaucescens TcmK(-) and TcmL(-) mutants and constructs harboring the tcmK and actI-ORF2 or actI-ORF1 and tcmL genes demonstrated that a heterologous pair of genes encoding a beta-ketoacyl acyl carrier protein synthase and chain length factor is often (but not always) nonfunctional. Isolations of the octaketides 6 and 7 from strains bearing actI-ORF1, actI-ORF2, and tcmM (pWHM766) or tcmJ, actI-ORF1, actI-ORF2, and tcmM (pWHM768), and the decaketides 8 and 9 from strains bearing tcmK, tcmL, and tcmM (pELE37) or tcmJ, tcmK, tcmL, and tcmM (pWHM731) established that the beta-ketoacyl acyl carrier protein synthase and chain length factor proteins determine the chain length of the polyketide. While the addition of the tcmJ gene to the polyketide-synthase core proteins consisting of the beta-ketoacyl acyl carrier protein synthase, chain length factor and acyl carrier protein had no affect on the structures of the resulting metabolites, adding the tcmN gene to either PWHM766 and pWHM768 or pELE37 and pWHM731 resulted in the synthesis of octaketide 5 or decaketide 2, respectively. TcmN thus can alter the regiospecificity for the first aldol cyclization from C-7/C-12 to C-9/C-14, suggesting that cyclases like TcmN determine the folding pattern of the linear polyketide intermediate. These activities, along with the choice of the starter unit, the loading of the extender unit to the PK complex, and the function of TcmJ are discussed in an attempt to provide a rationale for the engineered biosynthesis of novel polyketides.