The sesquiterpene lactone parthenolide is a promising anticancer drug. Its biosynthesis via a microbial cell factory has been considered as a sustainable alternative to plant extraction. Herein, systematic metabolic engineering approaches, as well as the introduction of a novel noncanonical tricarboxylic acid (TCA) cycle, were employed to enhance the production of the key precursor germacrene A. By identifying two new dehydrogenases and controlling the expression of parthenolide synthase, we further achieved the elimination of byproducts and enhanced parthenolide production. A two-stage fermentation approach and