Fig. 1: T5αH catalytic promiscuity is a bottleneck for pathway reconstitution and discovery.

a In the proposed biosynthetic pathway for paclitaxel, taxadiene synthase (TS) converts geranyl geranyl pyrophosphate (GGPP) to taxadiene (1), which can be oxidized to taxadien-5α-ol (2) by taxadiene 5α-hydroxylase (T5αH, in blue circle). Texts relevant to T5αH and the functional group installed by T5αH are highlighted in blue. Functional groups in the paclitaxel structures where biosynthetic enzymes are yet to be discovered are highlighted in red. The core 6/8/6-membered ring scaffold is highlighted with bold bonds. Picture of Taxus plants is taken by Jack C-T. Liu and used with permission. b GCMS total ion chromatograms (TICs) of Nicotiana benthamiana leaves transiently expressing cytosolic TS (cytoTS, black trace) or cytoTS and T5αΗ (blue trace) under 35S promoter in the pEAQ-HT vector²⁷. “Boost” indicates co-expression of truncated 3-hydroxy-3-methylglutaryl coenzyme-A reductase (tHMGR) and geranyl geranyl diphosphate synthase (GGPPS) to increase cytosolic diterpenoid yield as previously reported²⁸ and are always used unless otherwise noted. Compound 3 and 4 represent OCT and iso-OCT, respectively. TICs of N. benthamiana expressing GFP or boost alone (gray traces) are shown as control backgrounds. Compound 5–8 are structurally characterized in this study. Taxadiene (1) peak is filled black (baseline tilted to indicate the co-eluting peak) and oxidized taxadiene peaks are filled blue. “‡” indicates minor products generated by TS, including verticillene, taxa-4(20),11(12)-dien (iso-taxadiene), and taxa-3(4),11(12)-diene⁵⁴, whose identities are not assigned here. “*” indicates mono-oxidized taxadiene generated in N. benthamiana presumably by endogenous enzymes. The peak for geranylgeraniol (GGOH), an endogenously hydrolyzed side-product of GGPP, is also indicated. Representative traces of three biological replicates are shown.