Discovery of transporters in the plant natural product biosynthetic pathway for vinblastine

• Applicant: Dr. Ling Chuang
• Subject Area: Plant Biochemistry and Biophysics
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 559977563

Plant natural products (PNP) serve as valuable sources of essential medicines. The biosynthesis of PNPs exhibits complex spatial regulation with different steps of production and storage localized in specific subcellular, cellular, and tissue compartments. This necessitates the transport of intermediates and products across cell membranes, but the proteins that carry out the transport remain largely unknown. The proposed project aims at discovering and characterizing the transporters involved in the biosynthesis of the anticancer agent vinblastine, which is produced by the plant Madagascar periwinkle (Catharanthus roseus). Vinblastine has been approved for treating acute lymphocytic leukemia since the 1960s. Despite extensive synthetic and synthetic biology efforts, C. roseus remains the only known commercial source for vinblastine, and vinblastine shortages have been reported since 2019. The biosynthesis of vinblastine, the dimerization product of the monoterpene indole alkaloid (MIA) precursors catharanthine and vindoline, has been mostly elucidated. Yet the production of vinblastine based on synthetic biology approaches still results in low titers. The single-cell RNA sequencing data of C. roseus published last year reveals that vinblastine biosynthesis takes place in three distinct leaf cell types and pinpoints the intermediates that are likely to be transported. However, the mechanisms of translocation are still unknown. Understanding the transport processes in the native plant producers will provide the basis for transporter engineering to improve vinblastine production in heterologous hosts. This project will focus on discovering the transporters responsible for the intercellular movement of these molecules to provide a complete picture of how vinblastine intermediates are moved among these three cell types. I have identified promising transporter candidates from the published single-cell transcriptomic data via co-expression analysis. These candidates will be expressed and assayed with the predicted substrates using Xenopus oocytes, which have been established as a robust platform for expression of plant transporters. Transport activity into and out of oocytes will be assessed using mass spectrometry (LC-MS). The outcome of this project will not only build the foundation for optimizing the bioproduction of vinblastine but also other biosynthetically related MIAs that are pharmaceutically relevant. Moreover, since the transport mechanisms for many plant natural product biosynthetic pathways remain unknown, this work will provide fundamental insights into the broader area of PNP transport.

• DFG Programme: WBP Position