Isolation and functional characterization of terpene synthases from mediterranean plant Styrax officinalis

Terpenes are specialized metabolites widely distributed across life, including plants, bacteria, fungi, and insects, where they play diverse ecological and physiological roles. In plants, they contribute to defense, reproduction, and interactions with the environment. Styrax officinalis L. (Styracaceae) is an evergreen-deciduous tree or shrub that produces diverse specialized metabolites, including volatile terpenes of ecological and industrial importance. We used transcriptomic, metabolomic, and biochemical analyses to study S. officinalis, revealing the genetic and enzymatic mechanisms underlying terpene biosynthesis. RNA-seq analysis identified seven terpene synthases (StyTPS1-StyTPS7) and one putative acyclic terpene utilization protein (StyACTS). These genes were cloned, and their functions were experimentally confirmed. Transcript profiling revealed tissue-specific expression of StyTPS1 in flowers, StyTPS2-StyTPS5 in young leaves, and StyTPS6-StyTPS7 in mature leaves. Recombinant proteins with C-terminal His-tags were assayed using geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). Most TPS enzymes primarily produce acyclic monoterpenes, such as nerol (1). StyTPS3 and StyTPS4 showed substrate promiscuity, forming multiple mono- and sesquiterpenes. StyTPS5 produced defense-related sesquiterpenes, including (Z,E)-α-farnesene (2) and (E)-β-farnesene (3). StyTPS6 catalyzed the formation of diterpenes, including geranyllinalool (4), from GGPP. StyACTS produced linalool (5) and (Z)-nerolidol (6), representing novel functional terpene synthase-like activity in the plant system. The mono-, sesqui-, and diterpene products of StyTPS highlight substantial catalytic plasticity and reflect the evolutionary and ecological fitness of Styrax. These findings provide the first detailed functional map of terpene metabolism in S. officinalis and offer insights into terpene diversity, ecological roles, and biotechnological potential.