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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070852-03
Application #
7017030
Study Section
Special Emphasis Panel (ZGM1-MBP-9 (ME))
Program Officer
Jones, Warren
Project Start
2004-01-15
Project End
2008-12-31
Budget Start
2006-01-01
Budget End
2008-12-31
Support Year
3
Fiscal Year
2006
Total Cost
$230,319
Indirect Cost
Name
University of Massachusetts Amherst
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Patil, Rohan A; Lenka, Sangram K; Normanly, Jennifer et al. (2014) Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells. Plant Cell Rep 33:1479-92
Patil, Rohan A; Kolewe, Martin E; Roberts, Susan C (2013) Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures. Plant Cell Tissue Organ Cult 112:303-310
Lenka, Sangram K; Boutaoui, Nadia; Paulose, Bibin et al. (2012) Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells. BMC Genomics 13:148
Naill, Michael C; Kolewe, Martin E; Roberts, Susan C (2012) Paclitaxel uptake and transport in Taxus cell suspension cultures. Biochem Eng J 63:50-56
Patil, Rohan A; Kolewe, Martin E; Normanly, Jennifer et al. (2012) Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures. Biotechnol J 7:418-27
Kolewe, Martin E; Roberts, Susan C; Henson, Michael A (2012) A population balance equation model of aggregation dynamics in Taxus suspension cell cultures. Biotechnol Bioeng 109:472-82
McPartland, Timothy J; Patil, Rohan A; Malone, Michael F et al. (2012) Liquid-liquid extraction for recovery of paclitaxel from plant cell culture: solvent evaluation and use of extractants for partitioning and selectivity. Biotechnol Prog 28:990-7
Wilson, Sarah A; Roberts, Susan C (2012) Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol J 10:249-68
Stoppel, Whitney L; White, Joseph C; Horava, Sarena D et al. (2011) Transport of biological molecules in surfactant-alginate composite hydrogels. Acta Biomater 7:3988-98
Gaurav, Vishal; Roberts, Susan C (2011) Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates. Prep Biochem Biotechnol 41:219-35

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