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Abstract

The diterpenoid paclitaxel (Taxol®) is a chemotherapy medication widely used as a first-line treatment against several types of solid cancers. The supply of paclitaxel from natural sources is limited. However, missing knowledge of the genes involved in several specific metabolic steps of paclitaxel biosynthesis has rendered it difficult to engineer the full pathway. Here, we used a combination of transcriptomics, cell biology, metabolomics and pathway reconstitution to identify the complete gene set required for the heterologous production of taxol. We identified the missing steps from the current model of paclitaxel biosynthesis and confirmed the activity of most of the missing enzymes by heterologous expression in Nicotiana benthamiana. Notably, we identified a new C4β-C20 epoxidase which could overcome the first bottleneck of metabolic engineering. We used both previously characterized and newly identified oxomutase/epoxidase, taxane 1β-hydroxylase (T1βOH), taxane 9α-hydroxylase (T9αOH), taxane 9α-dioxygenase and phenylalanine-CoA ligase (PCL), to successfully biosynthesize the key intermediate baccatin III as well as for the conversion of baccatin III to paclitaxel in N. benthamiana. In combination, these approaches establish a metabolic route to taxoid biosynthesis and provide insights into the unique chemistry that plants use to generate complex bioactive metabolites.