The structural basis for the striking stereochemical discrimination among triketide analogs has been investigated by incubating a series of N-acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2+TE. The triketide analogs were first screened under a standard set of short-term incubation conditions in the presence of the extender substrate methylmalonyl-CoA and NADPH. For those triketide analogs that served as substrates for module 2+TE, the relative specificity, represented by the k(cat)/K(M) values, was quantitated. Triketide diastereomers that were converted in precursor-directed biosynthesis experiments to unsaturated 16-membered ring macrolides by DEBS(KS1(0)) were good to excellent substrates for DEBS module 2+TE, whereas analogs that were converted to the 14-membered ring analogs of 10,11-dehydro-6-deoxyerythronolide B by DEBS(KS1(0)) were not turned over at all by module 2+TE.