Bioorthogonal chemistry-driven multi-enzyme strategy for efficient terpenoid biosynthesis

Conventional in vivo terpenoid biosynthesis is often limited by the complexity of the intracellular environment and inefficient pathway flux. To overcome these challenges, an in vitro multi-enzyme cascade strategy was developed for the synthesis of high-value terpenoids. Using bioorthogonal protein pairing, six enzymes from the shortened mevalonate (MVA) pathway were assembled into ordered cross-linked enzyme complexes (O-CLEs), enabling controlled spatial organization. Multi-layer fluorescence labeling technology (MLFLT) confirmed the ordered arrangement of enzymes within the O-CLEs. For β-farnesene production, the assembled O-CLEs yielded 49.08 ± 1.91 mg/L of product, representing a 1.39-fold increase compared with the WT free-enzyme mixture (35.33 ± 0.51 mg/L) and a 3.03-fold increase compared with the (randomly cross-linked enzyme complexes) R-CLEs system 16.21 ± 1.68 mg/L), while retaining 62 ± 9% of initial activity after six successive reuse cycles. Notably, replacing β-farnesene synthase (FS) with taxadiene synthase (TXS) readily extended the strategy to taxadiene biosynthesis, demonstrating the modular design and general applicability of this approach. Thus, this bioorthogonal chemistry-driven, ordered multi-enzyme immobilization strategy offers a robust, generalizable, and reusable platform for the sustainable in vitro production of high-value terpenoids, advancing the development of in vitro synthetic biology.