Engineering a series of Scaffold-associated isoprenol utilization pathways to enhance terpene production spanning diverse chain lengths.

Terpenoids are valuable resources for pharmaceutical research, yet their natural supply remains constrained. While the artificial isoprenol utilization pathway (IUP) has emerged as a promising alternative for terpene precursor supply, its full potential is limited by suboptimal pathway flux and substrate tolerance. Here, we report an efficient scaffold-assisted IUP platform tailored for high-throughput terpenoid discovery and scalable production. By recruiting rate-limiting IUP enzymes and prenyltransferases (PTs) onto self-assembling PduA* protein scaffolds displaying CC-Di-B peptides that specifically interact with CC-Di-A-tagged enzymes, we achieved spatial organization of the biosynthetic machinery. Systematic optimization of promoter configuration, fermentation conditions, and enzyme fusion yielded the optimal system ScMKI4-GS, achieving gram-per-liter-scale production (1.1 g/L) of the eunicellane-type diterpene benditerpe-2,6,15-triene in simple shake-flask fermentation-substantially outperforming scaffold-free controls. The platform demonstrated broad applicability across five structurally distinct eunicellane synthases, with each exhibiting enhanced production upon scaffold incorporation. For lycopene biosynthesis, the scaffold-assisted system produced 729.7 mg/L-a 9-fold improvement over the canonical MVA pathway under identical conditions, representing the highest IUP-based lycopene titer reported in Escherichia coli to date. Finally, by systematic substitution of chain-length-specific PTs, we expanded the platform to efficiently generate C10-C35 terpene precursors, facilitating functional characterization of terpene-related genes. Collectively, this versatile scaffold-assisted IUP platform provides a robust tool to expand terpenoid structural diversity and accelerate their scalable overproduction.