Moving metabolites: Characterizing a MATE transporter in Madagascar periwinkle

Moving metabolites: Characterizing a MATE transporter in Madagascar periwinkle
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Plants are an essential reservoir for modern medicines, often serving as the source or inspiration for many pharmaceutical drugs. Perhaps one of the most significant examples is that of Madagascar periwinkle (Catharanthus roseus), a well-studied shrub which produces the life-saving anticancer drugs vincristine and vinblastine—used to treat a range of lymphomas and leukemias1. These compounds belong to a large class of plant specialized metabolites known as monoterpenoid indole alkaloids (MIAs). Their biosynthesis has been enzymatically resolved, culminating in its total reconstitution in yeast and other heterologous hosts2,3,4. Notably, decades of research5, including recent single-cell studies6,7,8, have revealed the highly compartmentalized nature of MIA biosynthesis in C. roseus, with modules or reaction steps restricted to specific organs (e.g., leaves), tissues (e.g., dermal), and multiple cell types (e.g., laticifers). This intricate metabolic patterning requires several transporters to move metabolites across the semi-permeable membranes that separate cells and subcellular organelles (e.g., the tonoplast).

In our study, we characterized CrMATE1, the first multi-antimicrobial extrusion protein (MATE) family transporter to have a role in MIA biosynthesis6,9. MATE family proteins are found across the kingdoms of life, transporting various low-molecular-weight compounds, often using proton or sodium antiport to energize translocation10. Since the discovery of the first MATE as a detoxifying efflux transporter in bacteria11, many have been characterized in plants, guiding the compartmentalization of specialized phenolic and alkaloid biosynthetic pathways12,13,14. However, CrMATE1 is unique in having a very narrow substrate preference for secologanin among the tested seco-iridoid metabolites9. The evolution of this specialized transport function appears to be conserved among closely related species within the Apocynaceae (dogbane) family.

Image of a Catharanthus flower adjacent to a schematic description of a simplified plant cell, with the molecules secologanin and tryptamine condensing to form strictosidine; a depiction of Xenopus laevis and the oocyte as a proxy for vacuolar transport.

Figure 1. Characterization of CrMATE1 as a vacuolar importer of secologanin in Catharanthus roseus using the Xenopus expression system. The transport of secologanin is required for the first committed step of monoterpenoid indole alkaloid biosynthesis, ultimately leading to the anticancer drug vinblastine.

In the context of MIA biosynthesis, the committing step into the pathway begins with the condensation of secologanin and tryptamine, forming strictosidine, the central intermediate potentiating a plethora of downstream metabolites (Figure 1). This reaction occurs in the vacuole of epidermis cells, requiring the transport of the components across the tonoplast. Transient expression of CrMATE1 in Nicotiana benthamiana confirmed tonoplast localization9. The importance of its function in maintaining secologanin flux into the MIA pathway was demonstrated by virus-induced silencing (VIGS), where CrMATE1-silenced plants developed a bottleneck affecting downstream MIAs. Secologanin appeared to build up in the cytosol, where it was reduced to the new metabolic intermediate secologanol.

Using the Xenopus laevis (African clawed frog) expression system (Figure 2), we conducted biochemical characterization of CrMATE1 to confirm its substrate range, directionality, and transport rate. CrMATE1 demonstrated strict directionality as a secologanin vacuolar importer (from neutral to acidic conditions)9. Furthermore, CrMATE1 rapidly translocated secologanin, moving 95% of the assayed substrate within 5 minutes of the assay.

Workflow of the Xenopus laevis expression system

Figure 2. Workflow of the Xenopus laevis expression system. CrMATE1 is cloned into an X. laevis expression vector and used as the template for the in vitro transcription of CrMATE1. The cRNA is microinjected into collagenase-treated oocytes, and maximal protein expression is reached in three days. The MATE-expressing oocytes are used in assays to test substrate import and export, with the pH of the media serving as a proxy for the acidic conditions of the plant vacuole.

The importance of studying the MIA pathway cannot be understated – C. roseus is the sole source of vinblastine and vincristine, and the current method of extraction of precursors from the plant requires 500 and 2000 kg of leaves to semi-synthesize 1g of the products, respectively3. The costly and inefficient reliance on plant cultivation and extraction has been a driving force behind MIA  research for over three decades. 

The “alternative supply chain” in engineered heterologous hosts, as mentioned above, has benefitted from this research; however, it is still far from approaching commercial viability at titers of 2.32 µg/L and 26.6 µg/L of the precursors vindoline and catharanthine, respectively3. Improving titers of high-value compounds requires a granular resolution of the pathway and identifying overlooked components, such as transporters, which can emulate, in part, the complex architecture that enables effective production in the plant.

Transporters can be a new tool for improving microbial yields by recovering substrates from the media and/or delivering flux between co-cultured stains. This utility has been demonstrated in attempts at reconstituting other specialized pathways, such as the engineering of yeast to produce the analgesic drugs codeine and morphine, with enzymes derived mainly from opium poppy (Papaver somniferum)15. In that report, deploying a purine permease-type uptake transporter in engineered yeast strains increased titers by 300-fold by recovering a broad range of benzylisoquinoline alkaloids (BIAs) and other intermediates lost to the media. Whether CrMATE1 or other MIA-associated transporters display similar properties has yet to be determined. The future for biotechnological applications of transporters in (re)engineering plants or heterologous hosts looks bright and can contribute to the development of biocatalytic manufacturing of medicinal MIAs.

 

References

  1. Courdavault, V., O’Connor, S.E., Oudin, A., Besseau, S., Papon, N. Towards the Microbial Production of Plant-Derived Anticancer Drugs. Trends in Cancer 6, 444-448 (2020).
  2. Dudley, Q.M. et al. Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana. Communications Biology 5, (2022).
  3. Zhang, J. et al. A microbial supply chain for production of the anticancer drug vinblastine. Nature 609, 341–347 (2022).
  4. Gao, J., Zuo, Y., Xiao, F. et al. Biosynthesis of catharanthine in engineered Pichia pastorisNat. Synth 2, 231–242 (2023).
  5. St-Pierre, B., Vazquez-Flota, F. A. & De Luca V. Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11, 887–900 (1999).
  6. Li, C. et al. Single-cell multi-omics in the medicinal plant Catharanthus roseus. Nat Chem Biol 19, 1031–1041 (2023).
  7. Guedes, J. G. et al. The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism. J Exp Bot 75, 274–299 (2024).
  8. Sun, S. et al. Single-cell RNA sequencing provides a high-resolution roadmap for understanding the multicellular compartmentation of specialized metabolism. Nature Plants 9, 179-190 (2022).
  9. Li, F. et al. Characterization of a vacuolar importer of secologanin in Catharanthus roseus. Commun Biol 7, 1–10 (2024).
  10. Upadhyay, N. et al. The multitasking abilities of MATE transporters in plants. Journal of Experimental Botany 70, 4643–4656 (2019).
  11. Morita, Y. et al. NorM, a Putative Multidrug Efflux Protein, of Vibrio parahaemolyticus and Its Homolog in Escherichia coli. Antimicrob Agents Chemother 42, 1778–1782 (1998).
  12. Morita, M. et al. Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc. Natl. Acad. Sci. U.S.A. 106, 2447–2452 (2009).
  13. Shoji, T. et al. Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots. Plant Physiology 149, 708–718 (2009).
  14. Ishimaru, Y. et al. A Rice Phenolic Efflux Transporter Is Essential for Solubilizing Precipitated Apoplasmic Iron in the Plant Stele. Journal of Biological Chemistry 286, 24649–24655 (2011).
  15. Dastmalchi, M. et al. Purine permease-type benzylisoquinoline alkaloid transporters in opium poppy. Plant Physiology 181, 916–933 (2019).

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Plant Molecular Biology
Life Sciences > Biological Sciences > Plant Science > Plant Molecular Biology
Plant Transporters
Life Sciences > Biological Sciences > Plant Science > Plant Cell Biology > Plant Transporters
Plant Secondary Metabolism
Life Sciences > Biological Sciences > Plant Science > Plant Secondary Metabolism
Transporters
Life Sciences > Biological Sciences > Molecular Biology > Protein Biochemistry > Proteins > Membrane Proteins > Transporters

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