(164ak) Functional Characterization of paclitaxel Transporters in Taxus Cuspidata to Enhance Secretion | AIChE

(164ak) Functional Characterization of paclitaxel Transporters in Taxus Cuspidata to Enhance Secretion

Authors 

Brzycki Newton, C., Worcester Polytechnic Institute
Revene, L., Worcester Polytechnic Institute
Roberts, S., University of Massachusetts, Amherst
Paclitaxel is a diterpenoid secondary metabolite naturally produced in Taxus species. The inherent medicinal activity of this drug has already been recognized and it has been widely used to treat various types of cancer. The advancement of plant cell culture technology has enabled industries to commercially produce paclitaxel to meet global demand. However, limited product storage capacity and the possibility of product degradation within plant cells limits overall yields. Manipulation of the paclitaxel biosynthetic and related pathways can be used to increase production. However, consideration of both transport and degradation can lead to superior strategies for yield improvements. Although the ABC transporter family is commonly responsible for exporting various plant secondary metabolites across the plasma membrane, no paclitaxel-specific transporters have yet been identified and characterized. In this study, putative paclitaxel transporter genes were identified by screening transcriptomic data for genes co-regulated with taxane biosynthesis and further comparing the homology of those genes with ABC transporters in other plants using BLASTP. From these data, we identified four uncharacterized proteins that display 70-80% homology for ABC-B or ABC-G transporters in related plant species. To characterize functional activity of these putative transporters, a knockout strain of S. cerevisiae, (AD1-8) which lacks all the essential ABC transporters in its genome was transformed with putative transporter genes following PEG-lithium-acetate based yeast transformation. The transgenic AD1-8 strain was then studied through the application of exogenous paclitaxel over time where extracellular and cell-associated paclitaxel levels were compared with that of the wild type (control) strain. A significant reduction in cell-associated paclitaxel concentration was observed in AD 1-8 expressing two of the four transporter genes, suggesting paclitaxel-specific transport activity of those putative genes. Efforts are now underway to overexpress relevant transporter genes in T. cuspidata cell lines to improve paclitaxel synthesis in culture through this novel strategy.