Integrated Platform for Structural and Functional Analysis of Cannabis sativa Terpene Synthases

Terpenoids are the largest and most diverse family of natural products. Volatile terpenes from Cannabis sativa are crucial in flavours, fragrances, and pharmaceuticals due to their unique odours and biological activities, including antimalarial, antibacterial, and insecticidal properties. Their synthesis is catalysed by terpene synthase (TPS) enzymes, which perform cyclisation and rearrangement reactions of over 55,000 distinct terpene compounds. However, low catalytic efficiency of C. sativa TPSs limits their use in large-scale commercial production. The complex biochemistry of these reactions is not well understood due to limited enzyme structure information. To address this, we developed a platform for recombinant expression, purification, enzymatic characterisation, and crystallisation of TPS enzymes from C. sativa. This includes comprehensive kinetic, thermostability, structural, and enzymatic analyses, along with a novel directed crystallisation screen informed by data mining existing conditions, facilitating diffraction-quality TPS crystals. Each TPS enzyme showed a distinct product profile, highlighting the need for systematic characterisation of C. sativa terpene biosynthesis.Our findings provide a framework for the structural and functional study of C. sativa TPSs. The developed platform sets the stage for future metabolic engineering aimed at optimising terpene production for pharmaceutical, pest management, and synthetic biology applications.