Releasing the potential power of terpene synthases by a robust precursor supply platform

Terpenoids represent the largest family of natural products. Their structural diversity is largely due to variable skeletons generated by terpene synthases. However, terpene skeletons found in nature are much more than those generated from known terpene synthases. Most promiscuous terpene synthases (i.e. those that can generate more than one product) have not been comprehensively characterised. Here, we first demonstrated that the promiscuous terpene synthases can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths (C₁₀, C₁₅, C₂₀, C₂₅). To release the synthetic potential of these enzymes, we integrated the engineered MVA pathway, combinatorial biosynthesis, and point mutagenesis to depict the comprehensive product profiles. In total, eight new terpenoids were characterised by NMR and three new skeletons were revealed. This work highlights the key role of metabolic engineering for natural product discovery.