Artificial Biosynthetic Pathways for Cell-Free Production of Raspberry Ketone and the Cardiotonic Drug Dobutamine

Abstract Raspberry ketone (RK) is a high-value flavor and fragrance compound widely used in the food, beverage, and cosmetic industries. However, existing biosynthetic routes largely rely on native metabolic pathways that involve multiple enzymatic steps and exhibit limited production efficiency. Here, we designed and constructed a retrosynthesis-guided artificial biosynthetic pathway for cell-free RK production. The pathway combines an artificial aldolase (ALD) with the native benzalacetone reductase (BAR) to convert 4-hydroxybenzaldehyde and acetone into RK through only two enzymatic steps. Following optimization of enzyme architecture and reaction conditions, the system produced 1.33 mM RK with a 66% conversion yield. We further extended this platform to the synthesis of the cardiotonic drug dobutamine through a non-natural reductive amination cascade using an engineered short-chain dehydrogenase/reductase (SDR). Rational protein engineering and byproduct recycling increased dobutamine production by approximately 104-fold relative to the wild-type enzyme. Both biosynthetic modules remained robust upon scale-up from microliter to milliliter volumes, and preparative-scale synthesis enabled isolation of 19.2 mg of RK. Overall, this work establishes artificial biosynthetic pathways for the production of a plant-derived natural product and a clinically relevant drug molecule, highlighting the potential of cell-free biomanufacturing as a sustainable platform for the synthesis of value-added chemicals and pharmaceuticals.