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.
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.
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
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