<jats:title>Significance</jats:title> <jats:p>Spliceostatins are bacterial natural products that show promising anticancer activity. Understanding how the bacterium makes spliceostatins will aid efforts toward a sustainable route for their production. Moreover, altering the chemical structure of a natural product is usually necessary to improve its pharmaceutical properties. For example, the parent spliceostatin molecule contains an unstable hemiketal chemical group. Contrary to previous hypotheses, we report on the identification of a dioxygenase enzyme responsible for hemiketal biosynthesis. Deletion of the corresponding dioxygenase gene led to a strain that produces exclusively spliceostatin congeners that are more stable than, and as active as, the parent compound, when derivatized to increase cell permeability. The strain generated in this study will be the basis for future development.