Jack Liu

All Publications

• Discovery of FoTO1 and Taxol genes enables biosynthesis of baccatin III. Nature McClune, C. J., Liu, J. C., Wick, C., De La Peña, R., Lange, B. M., Fordyce, P. M., Sattely, E. S. 2025

  Abstract

  Plants make complex and potent therapeutic molecules1,2, but sourcing these molecules from natural producers or through chemical synthesis is difficult, which limits their use in the clinic. A prominent example is the anti-cancer therapeutic paclitaxel (sold under the brand name Taxol), which is derived from yew trees (Taxus species)3. Identifying the full paclitaxel biosynthetic pathway would enable heterologous production of the drug, but this has yet to be achieved despite half a century of research4. Within Taxus' large, enzyme-rich genome5, we suspected that the paclitaxel pathway would be difficult to resolve using conventional RNA-sequencing and co-expression analyses. Here, to improve the resolution of transcriptional analysis for pathway identification, we developed a strategy we term multiplexed perturbation × single nuclei (mpXsn) to transcriptionally profile cell states spanning tissues, cell types, developmental stages and elicitation conditions. Our data show that paclitaxel biosynthetic genes segregate into distinct expression modules that suggest consecutive subpathways. These modules resolved seven new genes, allowing a de novo 17-gene biosynthesis and isolation of baccatin III, the industrial precursor to Taxol, in Nicotiana benthamiana leaves, at levels comparable with the natural abundance in Taxus needles. Notably, we found that a nuclear transport factor 2 (NTF2)-like protein, FoTO1, is crucial for promoting the formation of the desired product during the first oxidation, resolving a long-standing bottleneck in paclitaxel pathway reconstitution. Together with a new β-phenylalanine-CoA ligase, the eight genes discovered here enable the de novo biosynthesis of 3'-N-debenzoyl-2'-deoxypaclitaxel. More broadly, we establish a generalizable approach to efficiently scale the power of co-expression analysis to match the complexity of large, uncharacterized genomes, facilitating the discovery of high-value gene sets.

  View details for DOI 10.1038/s41586-025-09090-z

  View details for PubMedID 40500440

  View details for PubMedCentralID 7767101

• Multiplexed perturbation of yew reveals cryptic proteins that enable a total biosynthesis of baccatin III and Taxol precursors. bioRxiv : the preprint server for biology McClune, C. J., Liu, J. C., Wick, C., De La Peña, R., Lange, B. M., Fordyce, P. M., Sattely, E. S. 2024

  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®). 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 1β-hydroxylase (T1βH), taxane 9α-hydroxylase (T9αH), taxane 7β-O-acyltransferase (T7AT), taxane 7β-O-deacetylase (T7dA), taxane 9α-O-deacetylase (T9dA), and taxane 9-oxidase (T9ox). Importantly, the T9αH 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 β-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.

  View details for DOI 10.1101/2024.11.06.622305

  View details for PubMedID 39574719

  View details for PubMedCentralID PMC11580873

• Plants Utilize a Protection/Deprotection Strategy in Limonoid Biosynthesis: A "Missing Link" Carboxylesterase Boosts Yields and Provides Insights into Furan Formation. Journal of the American Chemical Society Hodgson, H., Stephenson, M. J., Kikuchi, S., Martin, L. B., Liu, J. C., Casson, R., Rejzek, M., Sattely, E. S., Osbourn, A. 2024

  Abstract

  The furan ring is a defining feature of limonoids, a class of highly rearranged and bioactive plant tetranortriterpenoids. We recently reported an apparent complete biosynthetic pathway to these important natural furanoids. Herein, we disclose the subsequent discovery of a yield-boosting "missing link" carboxylesterase that selectively deprotects a late-stage intermediate, so triggering more efficient furan biosynthesis. This has allowed, for the first time, the isolation and structural elucidation of unknown intermediates, refining our understanding of furan formation in limonoid biosynthesis.

  View details for DOI 10.1021/jacs.4c11213

  View details for PubMedID 39418479

• Reconstitution of early paclitaxel biosynthetic network. Nature communications Liu, J. C., De La Peña, R., Tocol, C., Sattely, E. S. 2024; 15 (1): 1419

  Abstract

  Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

  View details for DOI 10.1038/s41467-024-45574-8

  View details for PubMedID 38360800

  View details for PubMedCentralID PMC10869802

• Complex scaffold remodeling in plant triterpene biosynthesis. Science (New York, N.Y.) De La Pena, R., Hodgson, H., Liu, J. C., Stephenson, M. J., Martin, A. C., Owen, C., Harkess, A., Leebens-Mack, J., Jimenez, L. E., Osbourn, A., Sattely, E. S. 2023; 379 (6630): 361-368

  Abstract

  Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

  View details for DOI 10.1126/science.adf1017

  View details for PubMedID 36701471