Expression Profiling of Genes Involved in Biosynthesis of Paclitaxel (Taxol) and Characterization of Novel Transcripts Involved in the Defense Response

Ezekiel Nims (1), Nadia Boutaoui (1), Susan C. Roberts (1,2), and Elsbeth L. Walker (1,3)
(1) Plant Biology Graduate Program, University of Massachusetts, Amherst, MA 01003
(2) Department of Chemical Engineering, University of Massachusetts, Amherst,MA 01003
(3) Department of Biology, University of Massachusetts, Amherst, MA 01003

Taxol is a highly functionalized diterpenoid defense compound in the gymnosperm Taxus whose effective concentration in vivo is extremely low. This compound is used as an anti-cancer agent, but the supply is limiting, as these trees are slow growing and sparsely distributed.

Suspension cell culture offers an alternative to forest harvestation, but the accumulation of taxol is low and variable. Molecular characterization of this genus in regards to taxol biosynthesis and transport are key to understanding, and then alleviating this variable and limited taxol accumulation.

Here we show the expression profile of known genes in the biosynthetic pathway as a time course after elicitation using methyl jasmonate to instigate taxol production in cell culture. We show that there are potential bottlenecks in the terminal steps leading to taxol, where the expression of the two terminal genes is much lower than other pathway
enzyme transcripts. Taxane accumulation reflects this expression profile, where intermediates accumulate to high levels and taxol remains at low amounts.

Continuing work using subtractive libraries at three time points after elicitation is identifying novel transcripts involved in signaling, biosynthesis and transport of taxol or its intermediates. Validation of these libraries is underway using macroarray technology, and full-length clones of interest are being pulled out of cDNA libraries. These transcripts encode novel biosynthetic pathway enzymes and potential transporters of taxanes. Future work involves characterizing these genes and identifying targets for metabolic engineering to increase taxol production in suspension cell culture.