Identification and functional characterization of three monoterpene synthases from the medicinal plant Cinnamomum burmanii

Cinnamomum burmannii, a medicinal plant with a history of traditional use in Chinese folk medicine, utilizes various plant parts, including bark, root bark, leaves, and stems, for therapeutic purposes. Recent investigations into the essential oil extracted from the twigs and leaves of C. burmannii have revealed a notable abundance of volatile monoterpenes, especially D-borneol, α-pinene, and camphene. In this study, an extensive chemical profiling on the essential oil of the roots, stems, and leaves of C. burmannii was conducted. The analysis results indicated that the root and leaf components exhibit the most diverse and the most abundant of volatile monoterpenes, respectively. To elucidate the biosynthesis of monoterpenes in C. burmannii, candidate genes with monoterpene synthase activities were identified through transcriptome sequencing. Subsequently, function characterization on three mono-terpene synthases (TPSs), designated as CbTPS1, CbTPS2, and CbTPS3, were conducted using phylogenetic analysis and heterogeneous expression in Escherichia coli. The primary enzymatic products were identified as 3-carene, α-phellandrene, and bornyl diphosphate (BPP), respectively. Additionally, a confocal laser microscopy assay suggested the chloroplast localization of these mono-TPSs through transient expression in tobacco. Further validation of their functionality was confirmed through eukaryotic expression in tobacco. In conclusion, this study has unveiled critical enzymes responsible for the biosynthesis of major monoterpenes in C. burmannii. These findings provide essential elements for future studies in synthetic biology, facilitating a deep understanding of the biosynthetic pathways and potential applications in medicinal plant engineering.