Streamlined synthesis of β-elemene using a spatially organized multi-enzyme machinery

β-Elemene is a pharmaceutically important sesquiterpene, while its production from plants and chemical synthesis remains inefficient. To achieve the efficient in vitro synthesis of β-elemene from low-cost mevalonate, a hexa-enzyme cascade system was employed to improve biosynthetic productivity. Here we show that orderly cross-linked hexa-enzyme complexes constructed via bio-orthogonal protein pairs (Tag/Catcher, RIAD/RIDD₂, and K/Q-Tag) enable spontaneous one-step co-purification and immobilization of six enzymes for β-elemene synthesis. These organized assemblies, confirmed for their spatial co-localization, achieve a β-elemene titer of 160.08 mg/L, a substantial improvement over the 27.54 mg/L produced by the corresponding free multi-enzyme system. In addition, the cross-linked complexes combine favorable operational stability with facile centrifugation-based recovery, retaining approximately 70% activity over six reuse cycles. Bio-orthogonal multi-enzyme assembly engineering to obtain complexes enables efficient in vitro biosynthesis of β-elemene, guided by nature's metabolic synthesis. This strategy exploits the mevalonate pathway's utility for sustainable engineered synthesis of terpenoid compounds.