Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Characterization of O-methyltransferases involved in the biosynthesis of tetrandrine in Stephania tetrandra.

Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.

Transcriptomic Insight into Terpenoid Biosynthesis and Functional Characterization of Three Diterpene Synthases in Scutellaria barbata.

Scutellaria barbata (Lamiaceae) is an important medicinal herb widely used in China, Korea, India, and other Asian countries. Neo-clerodane diterpenoids are the largest known group of Scutellaria diterpenoids and show promising cytotoxic activity against several cancer cell lines. Here, Illumina-based deep transcriptome analysis of flowers, the aerial parts (leaf and stem), and roots of S. barbata was used to explore terpenoid-related genes. In total, 121,958,564 clean RNA-sequence reads were assembled into 88,980 transcripts, with an average length of 1370 nt and N50 length of 2144 nt, indicating high assembly quality. We identified nearly all known terpenoid-related genes (33 genes) involved in biosynthesis of the terpenoid backbone and 14 terpene synthase genes which generate skeletons for different terpenoids. Three full length diterpene synthase genes were functionally identified using an in vitro assay. SbTPS8 and SbTPS9 were identified as normal-CPP and ent-CPP synthase, respectively. SbTPS12 reacts with SbTPS8 to produce miltiradiene. Furthermore, SbTPS12 was proven to be a less promiscuous class I diterpene synthase. These results give a comprehensive understanding of the terpenoid biosynthesis in S. barbata and provide useful information for enhancing the production of bioactive neo-clerodane diterpenoids through genetic engineering.

Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens.

Ent-kaurene diterpenoids are the largest group of known Isodon diterpenoids. Among them, oridonin is accumulated in the leaves, and is the most frequently studied compound because of its antitumor and antibacterial activities. We have identified five copalyl diphosphate synthase (CPS) and six kaurene synthase-like (KSL) genes by transcriptome profiling of Isodon rubescens leaves. An in vitro assay assigns ten of them to five different diterpene biosynthesis pathways, except IrCPS3 that has a mutation in the catalytic motif. The Lamiaceae-specific clade genes (IrCPS1 and IrCPS2) synthesize the intermediate copalyl diphosphate (normal-CPP), while IrCPS4 and IrCPS5 synthesize the intermediate ent-copalyl diphosphate (ent-CPP). IrKSL2, IrKSL4, and IrKSL5 react with ent-CPP to produce an ent-isopimaradiene-like compound, ent-atiserene and ent-kaurene, respectively. Correspondingly, the Lamiaceae-specific clade genes IrKSL1 or IrKSL3 combined with normal-CPP led to the formation of miltiradiene. The compound then underwent aromatization and oxidization with a cytochrome P450 forming two related compounds, abietatriene and ferruginol, which were detected in the root bark. IrKSL6 reacts with normal-CPP to produce isopimaradiene. IrKSL3 and IrKSL6 have the γßα tridomain structure, as these proteins tend to possess the bidomain structure of IrKSL1, highlighting the evolutionary history of KSL gene domain loss and further elucidating chemical diversity evolution from a macroevolutionary stance in Lamiaceae.