Multiplexed perturbation reveals cryptic proteins that enable a total biosynthesis of baccatin III and Taxol precursors
Plants make complex and potent therapeutic molecules, but difficulties in sourcing from natural producers or chemical synthesis can challenge their use in the clinic. A prominent example is the anti-cancer therapeutic paclitaxel (Taxol®) from yew (Taxus) trees. Identification of the full paclitaxel biosynthetic pathway would enable heterologous drug production, but it has eluded discovery despite a half century of intensive research. Within the search space of Taxus' large, enzyme-rich genome, we suspected the complex paclitaxel pathway would be difficult to resolve using conventional gene co-expression analysis and small sample sets. To improve the resolution of gene set identification, we developed a multiplexed perturbation strategy to transcriptionally profile cell states spanning tissues, cell types, developmental stages, and elicitation conditions. This approach revealed a set of paclitaxel biosynthetic genes that segregate into expression modules that suggest consecutive biosynthetic sub-pathways. These modules resolved seven new genes that, when combined with previously known enzymes, are sufficient for the de novo biosynthesis and isolation of baccatin III, an industrial precursor for Taxol, in Nicotiana benthamiana leaves at levels comparable to the natural abundance in Taxus needles. Included are taxane 1ß-hydroxylase (T1ßH), taxane 9a-hydroxylase (T9aH), taxane 7ß-O-acyltransferase (T7??), taxane 7ß-O-deacetylase (T7dA), taxane 9a-O-deacetylase (T9dA), and taxane 9-oxidase (T9ox). Importantly, the T9aH we discovered is distinct and independently evolved from those recently reported, which failed to yield baccatin III with downstream enzymes. Unexpectedly, we also found a nuclear transport factor 2 (NTF2)-like protein (FoTO1) crucial for high yields of taxanes; this protein promotes the formation of the desired product during the first taxane oxidation step, resolving a longstanding bottleneck in paclitaxel pathway reconstitution. Together with a new ß-phenylalanine-CoA-ligase, the eight genes discovered in this study enables the complete reconstitution of 3'-N-debenzoyl-2'-deoxy-paclitaxel with a 20-enzyme pathway in Nicotiana plants. More broadly, we establish a generalizable approach for pathway discovery that scales the power of co-expression studies to match the complexity of specialized metabolism, enabling discovery of gene sets responsible for high-value biological functions. Overall design: To improve the resolution of gene set identification, we developed a multiplexed perturbation strategy to transcriptionally profile cell states spanning tissues, cell types, developmental stages, and elicitation conditions. To maximize the probability of activating biosynthetic states, we compiled a panel of elicitors including plant hormones (associated with growth, abiotic stress, and biotic stress), pathogen associated molecular patterns (PAMPs), taxane pathway intermediates, and select bacterial and fungal species. This approach revealed a set of paclitaxel biosynthetic genes that segregate into expression modules that suggest consecutive biosynthetic sub-pathways.