Engineering Protein Scaffold-Assisted C10-C35 Terpene Precursors Overproduction System for Efficient Biosynthesis Studies

Terpenoids are widely used in the food, cosmetics, and pharmaceutical fields. However, the application is hindered by insufficient supplies of natural origin. The microbial biosynthesis presents an alternative to conventional techniques. However, its efficiency remains hampered by two key factors: an inadequate supply of precursors and the lack of a versatile production platform. Here, an efficient scaffold-associated system is developed for efficient discovery and production of terpenoids. A self-assembling scaffold is employed to recruit enzymes of the precursor pathway, thereby enhancing precursor synthesis. This scaffolded upstream module not only improves substrate tolerance but also significantly boosts catalytic efficiency, resulting in gram-per-liter-scale production of a eunicellane diterpene with simple shake-flask fermentation. The compatibility of the system is verified by introducing multi-enzyme pathways, yielding high-level production of lycopene with a relatively 9-fold higher efficiency than the MVA pathway. Furthermore, the platform is extended to generate a range of C10–C35 terpene precursors, facilitating the characterization of various prenyltransferases and terpene synthases for terpene skeletons expansion. Notably, it enables the discovery of EcTPS2, which represents the first non-squalene triterpene synthase of animal origin. This system offers a versatile and highly efficient platform for expanding the diversity and enhancing the overproduction of terpenoids.