Fig. 2: Characterization of T5αΗ product profile.

a Synthesis of taxadien-5α-ol (2). Star indicates the structure is confirmed by NMR in this study. b GCMS mass spectra of 2 originated from chemical synthesis (magenta) and heterologous production in N. benthamiana (blue). c Structures of T5αH products when T5αH is heterologously expressed in N. benthamiana. Star indicates the structure is confirmed by NMR in this study. Oxidative modifications are shown in red. d GCMS total ion chromatograms (TICs) of Nicotiana benthamiana leaves transiently expressing GFP or T5αH with synthetic 2 fed into leaves three days post infiltration. Compound 9 is a di-oxidized taxadiene whose structure is not characterized. e GCMS TICs of Nicotiana benthamiana leaves transiently expressing cytosolic diterpenoid boosting enzymes (boost) previously reported²⁸, cytosolic TS (cytoTS), T5αH and taxadien-5α-ol O-acetyltransferase (TAT). Two acetylated product 10 and 11 were observed with concurrent decrease of compound 2 and 9, illustrating how the complexity of the product profile increases with additional downstream enzymes when T5αH side-products are present in the reconstitution system. Products of T5αH are colored red and those of TAT are colored orange.