Plant cell cultures can be used to alleviate the demands placed on harvest for the supply of important plant-derived medicinals. Due to economic constraints, there are currently few plant cell culture processes used commercially for the manufacture of such products. Limitations in low volumetric productivity and variability in productivity can be overcome through a better understanding of global metabolic control. Plant cell culture has received significant attention as a supply method for the anti-cancer agent paclitaxel, which has been approved by the FDA for the treatment of breast, ovarian and lung cancers as well as the AIDS-related Kaposi's sarcoma. The proposed research project centers on the use of molecular approaches to delineate global metabolic control of paclitaxel accumulation in Taxus cell cultures. We propose a significantly different approach than other research groups, one that uses transcription profiling to identify genes involved in global metabolic control. Such genes will be involved not just in paclitaxel biosynthesis (where all current efforts are focused), but also in transcriptional regulation, transport, secretion and degradation.
The specific aims of this study are to (1) identify changes in gene expression in Taxus suspension cultures with enhanced accumulation of paclitaxel through the identification of mRNAs that accumulate differentially, (2) construct high quality cDNA libraries and isolate full-length clones of these differentially expressed mRNAs, (3) predict putative gene function using publicly available bioinformatics resources, and (4) functionally characterize differentially expressed genes using both stable transformation technologies and assaying of relevant enzymatic activities. The results of the proposed research will identify genes involved in global control of paclitaxel metabolism which can then be used in the design of strategies to optimize the plant cell culture process for paclitaxel production and help alleviate the paclitaxel supply problem for both patient treatments and clinical trials. It is important to emphasize that the outcomes of the proposed research, particularly the work centering around methods development, has the potential to significantly impact research on the production of other valuable medicinals from plant cell culture systems.
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