Nicotiana benthamiana, Domin

Andersen, Trine Bundgaard, Rasmussen, Silas Anselm, Christensen, Søren Brøgger & Simonsen, Henrik Toft, 2019, Biosynthesis of tovarol and other sesquiterpenoids in Thapsia laciniata Rouy, Phytochemistry 157, pp. 168-174 : 169

2.2. Characterization of TlTPS820 expressed in N. benthamiana

Leaves from N. benthamiana expressing Tl TPS820 were extracted with hexane and analyzed by GC-MS. The analysis yielded a sesquiterpenoid profile identical to Tg TPS2 ( Fig. 1 ). The main product of Tg TPS2 is epikunzeaol (1) ( Fig. 2 ), thus Tl TPS820 is also an epikunzeaol synthase and an orthologue of Tg TPS2. As described previously, the high injection port temperature at 250 ̊C lead to cope-rearrangement of 1 ( Andersen et al., 2017). Thus, 1 was partly degraded to two unknown alcohols.

Figures

Fig. 1. GC-MS chromatogram of hexane extracts from N. benthamiana expressing AttHMGR+ TlTPS820, AttHMGR+TgTPS2, and AtHMGR + TlTPS820 + TlCYP76AE4. The chromatograms show that TgTPS2 and TlTPS820 both produce epikunzeaol (1) as previously published for TgTPS2 (Andersen et al., 2017), and with co-expression of TlCYP76AE4 this product disappear and a cope rearranged product of tovarol (2) is found. This confirms the functionality of both enzymes.

Fig. 2. The biosynthetic routes catalyzed by the four described Thapsia laciniata enzymes TlTPS820, TlTPS509, TlTPS18983, and TlCYP76AE4 leading to tovarol (2).