(160am) Transcriptome-Guided Mining and Functional Characterization of paclitaxel Transporters in Taxus Plant Cell Culture

Plant-derived secondary metabolites have many applications as fragrances, food additives, dyes, and bioactive medicinal compounds. Many medicinal plants have developed unique biosynthetic capabilities that makes production of these metabolites in plant cell culture (PCC) an attractive alternative to extensive engineering of model hosts. While pathway engineering approaches have been moderately successful at increasing production of secondary metabolites in PCC, intracellular trafficking and export of product from the cell is known to be a rate-limiting step in many PCC production processes. This is particularly true for paclitaxel, a chemotherapeutic drug produced industrially using Taxus PCC. Despite evidence that paclitaxel export in Taxus is likely regulated by one or more ATP-binding cassette (ABC) transporters, which are often responsible for transport of plant secondary metabolites across the plasma membrane, no paclitaxel-specific transporters have yet been characterized. Fundamental insights into regulation of paclitaxel localization and transport are therefore necessary to overcome transport limitations and optimize product yield. Here, we present our work on transcriptome-guided mining and functional characterization of putative paclitaxel transporters in ABC transporter-deficient S. cerevisiae strain AD 1-8.

To more specifically elucidate the mechanism of paclitaxel transport in Taxus PCC, Taxus canadensis cultures were spiked with exogenous paclitaxel and treated with a variety of different ABC transport inhibitors with specificities for different families of transporters. Treatment with verapamil and genestein (both known ABC-B and ABC-G transport inhibitors) resulted in higher concentrations of intracellular paclitaxel and lower concentrations of extracellular paclitaxel compared to untreated cultures, indicating inhibition of paclitaxel export. This suggests that any putative paclitaxel transporters would likely belong to the ABC-B or ABC-G transporter families.

Differential transcriptome analysis of Taxus cuspidata cultures in response to elicitation with methyl jasmonate, a well-characterized activator of secondary metabolism in numerous plant systems, was used to identify genes associated with metabolic regulation and cellular trafficking of paclitaxel. From these data, we identified 4 uncharacterized proteins that display 70-80% homology for ABC-B or ABC-G transporters in related plant species. Transmembrane helix prediction software confirms that these uncharacterized proteins contain transmembrane domains and nucleotide binding domains consistent with those of either ABC-B or ABC-G transport families.

To characterize the functional activity of these putative transporters, we constitutively expressed them in an ABC-transporter deficient strain of S. cerevisiae (AD 1-8), which is deficient in all essential ABC transporters and has previously been used successfully for heterologous characterization of plant transport proteins. Uptake and export of Flutax-2, a fluorescent paclitaxel analog, was measured over time for strains expressing each putative transporter and compared to an empty vector control. To evaluate substrate specificity, cultures were saturated with paclitaxel or precursors 10-deacetylbaccatin (10-DAB) or baccatin III to determine which taxanes competitively inhibit transport of Flutax-2. By characterizing the functionality of these putative paclitaxel transporters, we can ultimately develop novel strategies for engineering cellular transport in Taxus plant cell culture.