Polyketides represent a diverse family of natural products that are synthesized by three major types of polyketide synthases (PKSs) using simple acyl-CoA substrates. How iterative Type I PKSs, specifically, are programmed to select starter units, extend intermediates to a particular chain length, control cyclization chemistry, and release products are unanswered central questions in natural products chemistry. This thesis addresses the intriguing enzymology of a fungal Type I iterative fatty acid synthase (FAS)/PKS complex involved in the production of norsolorinic acid, a polycyclic ketide intermediate at the start of the biosynthetic pathway to the potent environmental pro-hepatocarcinogen, aflatoxin. Mono-, di-, and tridomains from the FAS/PKS complex were independently cloned, expressed in E. coli, and purified to assess their enzymatic functions. After assaying individual components, the parts were reconstituted in combinatorial experiments to analyze the assembly steps that lead to product formation. This dissection/reassembly process, "deconstruction," allows one to probe mechanistic questions hidden by the nature of multidomainal iterative enzymes where no free intermediates are released. Full polyketide processing was carried out by multi-part assemblies of these constructs, demonstrating that necessary inter-domainal interactions occured whether they are tethered to each other or not. While employing this conceptual approach, a previously unrecognized region in the PKS was discovered to be a starter unit:acyl-carrier protein transacylase domain that selects a C₆-fatty acid starter from the associated FAS and transfers it on to the PKS ACP to initiate polyketide biosynthesis. Search of protein databases revealed that SAT domains were widespread among "nonreducing" fungal PKSs. Homologous PKS SAT domains exhibited acetyl specificity when not associated with a FAS, providing a biochemical explanation for the observation of the classical "starter unit effect" in fungal polyketide biosynthesis. These combinatorial enzymology experiments also led to the discovery of a second unknown domain, which we have dubbed the "product template." This domain controls the cyclization/dehydration chemistry of the elongated poly-keto intermediate. Two new catalytic domains were discovered using this approach, and insights have been gained into the factors that control chain length, cyclization control, and product release for fungal aromatic polyketides. This work also has led to fruitful collaborations. The crystal structures for the product template and the thioesterase/Claisen cyclase domains, which control cyclization and product release, respectively, were determined, and the masses of intermediates attached to the enzyme during polyketide processing were measured, revealing further mechanistic insight into iterative enzymatic catalysis.