The rubromycins, such as gamma-rubromycin, heliquinomycin, and griseorhodin A, are a family of extensively modified aromatic polyketides that inhibit HIV reverse transcriptase and human telomerase. Telomerase inhibition crucially depends on the presence of a spiroketal moiety that is unique among aromatic polyketides. Biosynthetic incorporation of this pharmacophore into the rubromycins results in a dramatic distortion of the overall polyketide structure, but how this process is achieved by the cell has been obscure. To identify the enzymes involved in spiroketal construction, we generated 14 gene-deletion variants of the griseorhodin A biosynthetic gene cluster isolated from the tunicate-associated bacterium Streptomyces sp. JP95. Heterologous expression and metabolic analysis allowed for an assignment of most genes to various stages of griseorhodin tailoring and pharmacophore generation. The isolation of the novel advanced intermediate lenticulone, which exhibits cytotoxic, antibacterial, and elastase-inhibiting activity, provided direct evidence that the spiroketal is formed by cleavage of four carbon-carbon bonds in a pentangular polyketide precursor. This remarkable transformation is followed by an epoxidation catalyzed by an unusual cytochrome P450/NADPH:ubiquinone oxidoreductase pair that utilizes a saturated substrate. In addition, the absolute configuration of griseorhodin A was determined by quantum-chemical circular dichroism (CD) calculations in combination with experimental CD measurements.