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BiochemistryNature
Peer-reviewed journal article

Multiplexed perturbation of yew reveals cryptic proteins that enable a total biosynthesis of baccatin III and Taxol precursors

Conor James McCluneiD, Chun‐Ting LiuiD, Chloe WickiD, Ricardo De La PeñaiD, Bernd Markus LangeiD, Polly Morrell FordyceiD, Elizabeth S. SattelyiD

Abstract

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(R)). 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 1{beta}-hydroxylase (T1{beta}H), taxane 9-hydroxylase (T9H), taxane 7{beta}-O-acyltransferase (T7AT), taxane 7{beta}-O-deacetylase (T7dA), taxane 9-O-deacetylase (T9dA), and taxane 9-oxidase (T9ox). Importantly, the T9H 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 gene 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 {beta}-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.

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In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (16 Nov 2024) · CC BY 4.0

A multiplexed perturbation and single-nucleus RNA-seq screen of Taxus needles found eight new Taxol pathway genes, including FoTO1, enabling baccatin III and a late Taxol precursor in N. benthamiana.

The Taxol pathway had resisted discovery, and bulk co-expression in Taxus lacked the resolution to separate Taxol genes from the many other specialized-metabolism genes. The paper reports a perturbation-multiplexed snRNA-seq design that ranks the new genes far higher than bulk RNA-seq, and it completes a 17-gene baccatin III route plus a 20-gene route to 3'-N-debenzoyl-2'-deoxypaclitaxel.

Key results

  1. ShownPooling 17,143 single-nucleus profiles into pseudobulk cell states from many perturbations resolved Taxol genes better than public bulk RNA-seq. All but two of 14 known Taxol genes ranked higher by correlation to TDS.

    mpXsn: 2,901 cell states from 17,143 profiles (272 elicited tissue samples) vs 79 bulk samples from six studies. The eight new genes ranked about 1,000th-10,000th with bulk data but within the top 10s-100s with mpXsn (T9dA excepted, per the figure legend). · Multiplexed chemical elicitation produces cells…; Expression profile and essentiality of the new…; Multiplexed tissue elicitation; Figure legends

  2. ShownConsensus non-negative matrix factorization of the gene-by-cell matrix split Taxol genes into three expression modules that suggest consecutive sub-pathways. Module 1 holds the early pathway and is linked to the MEP pathway, and elicitation was needed to activate it.

    3 of 200 modules were enriched in known Taxol enzymes; no single module had all known enzymes in its top 200. The three Taxol modules ranked first for enzyme count in the top 100 genes. · Identification of three Taxol biosynthetic gene…

  3. ShownThe NTF2-like protein FoTO1 redirects T5αH oxidation away from rearranged side products toward taxadien-5α-ol when expressed in N. benthamiana. Binding and co-IP data suggest direct interaction with T5αH, but the mechanism is not resolved.

    10-17 fold higher yields from early subpathways (n = 3 leaves, Welch's t-test); side products 2'a-c no longer detected. Microscale thermophoresis showed high-nanomolar KD for T5αH and TDS2, lost on C-terminal helix deletion. T5αH co-IP rose four-fold with 400 μM taxadiene. · Discovery of Facilitator of Taxane Oxidization…; DISCUSSION; Figure legends

  4. ShownBeyond FoTO1, new genes T9αH-750C, T7AT, T1βH-686 (plus related T1βH-184), T7dA, T9dA, T9ox and the CoA-ligase PCL complete the pathway (eight new genes with FoTO1). This gives a 17-gene route to baccatin III and a 20-enzyme route to 3'-N-debenzoyl-2'-deoxypaclitaxel in N. benthamiana; Taxol was not reached.

    Baccatin III identified by NMR and MS/MS vs a standard; yield 10-30 ug/g, equivalent to Taxus media needles. Published gene sets gave no baccatin III above the detection limit in this system. Single-gene dropouts abolished production except FoTO, T5αH, DBAT and T9ox. · Discovery of two taxane deacetylases and the…; Side-chain biosynthetic enzymes enable total…; Expression profile and essentiality of the new…; DISCUSSION

  5. ShownThe new T9αH (CYP750C family) is unrelated in sequence to the recently reported CYP725A T9αH and differs in substrate preference. Swapping in the CYP725A enzyme gave no detectable baccatin III in this pathway.

    Under 20% protein identity between T9αH-750C and T9αH-725A; only T9αH-750C depleted the 3O2A intermediate to give 4O3A, while T9αH-725A appeared to need a C-13α acetoxy substrate. · Independently evolved taxane 9α-hydroxylases…; Expression profile and essentiality of the new…

Methods, data and reproducibility

Needles from Taxus media were exposed to a panel of elicitors and sampled over 1-4 days. Their nuclei were pooled into one snRNA-seq library, and co-expression and cNMF modules were used to rank candidate genes. Candidates were tested by transient co-expression in N. benthamiana with LC-MS/GC-MS, NMR, binding and co-IP assays.

  • snRNA-seq of Taxus media needles: elicited (8,039), naive young (3,027) and naive mature (6,077) profiles, 17,143 after integration · Taxus media · n = 17,143 profiles; 2,901 cell states; 272 tissue samples
  • Six previously published bulk RNA-seq studies of Taxus, mapped to the T. chinensis genome · Taxus · n = 79 samples
  • Taxus media genome used for BLAST search of gene models · Taxus media · NCBI PRJNA1136025
  • Transient expression of Taxus genes in leaves, with GC-MS/LC-MS quantification · Nicotiana benthamiana · n = n = 3 biological leaf replicates per condition (n = 6 for one comparison)
  • NMR of isolated taxanes (taxusin, 13β-taxusin, 1β-hydroxytaxusin, 15-hydroxy-11(15→1)abeo-taxusin) · Nicotiana benthamiana (expression host) · n = 4 compounds · NP0341906, NP0341907, NP0341908, NP0341909
Methods & tools
STARsolo, CellBender, DoubletDetection, scVI, Scanpy, consensus NMF (cNMF), AlphaFold3, Clustal Omega, Geneious Prime, GC-MS, LC-MS (Q-TOF and triple quadrupole), microscale thermophoresis
Data & accessions
http://np-mrd.org/
Reagents & environment
Geneious Prime v2024.0.4 and MestReNova v14.3.1 are named; no container or requirements file is mentioned.
Reproducibility gaps
No code repository is given. No accession is printed for the new snRNA-seq data. Gene lists, primers and supplementary tables are referenced but not in the text. Only the NMR raw data are deposited.
Limitations (4)
  • Taxol itself was not produced: T2'αH could not be made to work, and DBTNBT gave only very low-level 2'-deoxypaclitaxel, so the pathway ends at 3'-N-debenzoyl-2'-deoxypaclitaxel. · stated by the authors
  • The heterologous route runs via C-13α-hydroxyl intermediates, and it is not known whether Taxus uses the same route. No C-13α-O-deacetylase was found to access baccatin III through C-13α-acetoxy intermediates. · stated by the authors
  • The mechanism of FoTO1 is unresolved. Its homologs from T. media and Arabidopsis did not reproduce the effect, and the authors urge caution in extrapolating its function. · stated by the authors
  • Gene function was tested only by transient expression in N. benthamiana leaves with three biological replicates, with no knockout or silencing in Taxus. Failure to reproduce published gene sets may reflect differences in this system. · noted in this summary

Who should read:Read this if you work on plant specialized-metabolism gene discovery, single-nucleus co-expression in non-model species, or heterologous Taxol and baccatin III production.

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