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. Terpenoids, with their diverse structures and significant pharmaceutical and industrial applications, face production challenges due to inefficient plant extraction and synthesis methods. Here, the authors explore microbial engineering strategies to enhance terpenoid production, leveraging enzyme complexes for improved substrate channeling.