Fig. 5: Metabolic network of early paclitaxel biosynthesis.

GCMS total ion chromatograms (TICs) and LCMS extracted ion chromatograms (EICs) of plant extracts where taxadien-5α-ol (2) is fed into N. benthamiana leaves expressing different combinations of paclitaxel biosynthetic genes to examine all possible pathways. Taxadiene 5α-hydroxylase (T5αH) and its product are colored red; taxadien-5α-ol O-acetyltransferase (TAT) and its product are colored orange; taxane 10β-hydroxylase (T10βΗ) and its product are colored green; 10-deacetylbaccatin III:10-O-acetyltransferase (DBAT) and its product are colored blue; taxane 13α-hydroxylase (T13αΗ) and its product are colored purple. Cytochrome P450 T10βH and T13αH are expressed under the NOS promoter, and all other genes are expressed under the 35S promoter. A retention time window of 0.5 min is shown for LCMS EIC. The proposed metabolic network and intermediate structures are shown. Structures 16–21 are proposed based on the characterized function of each enzyme in this work and the literature^22,23,24,25 as well as analysis of their mass spectra (Supplementary Table 2). “Taxadien” is omitted in the names of most compounds for simplicity. Dash arrow indicates reaction by T13αH that could potentially be involved in the metabolic network but for which we do not have direct evidence. Asterisk indicates co-eluting compounds not related to taxanes. The peak for geranylgeraniol (GGOH), an endogenously hydrolyzed side-product of GGPP, is also indicated.