To apply metabolic engineering strategies to improve production of the anti-cancer agent paclitaxel in Taxus plant cell cultures

	Examine expression profiles of known taxane biosynthetic pathway genes and correlate to metabolite analysis to identify potential rate-limiting steps in paclitaxel accumulation
	Apply transcript profiling to identify global changes in gene expression upon enhanced paclitaxel accumulation induced by methyl jasmonate elicitation
	Construct high quality cDNA libraries and isolate full-length clones of differentially expressed mRNAs
	Assign putative functions for metabolic control genes using publicly available bioinformatics resources
	Develop a transformation protocol for Taxus plant suspension cultures
	Stably transform Taxus cell lines that overexpress selected cDNAs and perform detailed metabolite analyses and characterization studies

	Suppression subtractive hybridization has been successfully optimized and applied to generate over 200 clones that are differentially expressed in methyl jasmonate-elicited and control cultures
	Hybridization probes for each known gene in the paclitaxel biosynthetic pathway have been generated and patterns of gene expression in methyl jasmonate-elicited and control cultures have been identified; rate-limiting steps have been proposed
	An Agrobacterium-mediated transformation protocol has been developed for Taxus suspension cultures and is currently being optimized

	Improve production of valuable medicinals such as paclitaxel from plant cell cultures
	Develop a novel approach for the targeted application of metabolic engineering to plant organisms with unsequenced genomes and minimal genetic characterization
	Create transgenic cell lines specifically for use in paclitaxel production processes

Abstract:

Metabolic engineering research has largely focused on microbes, with an emerging emphasis on plants and animals.  Plants are critically important to the world’s food resource, synthesis of bio-based goods (e.g., chemicals, energy, etc), and supply of pharmaceutically active products.  Metabolic engineering of plant cell systems offers distinct challenges over microbial systems including slow plant growth, metabolic compartmentalization and redundancy, and lack of genetic characterization.  There have been some recent successes in the reconstitution of plant biosynthetic pathways in fast-growing microbes, but because of the complexity of many plant products, transformation of entire pathways is often not possible.  Therefore, the application of systems-level approaches to characterizing plant metabolism is necessary in the design of optimal plant-based systems, including those centered on plant cell culture. 

The use of plant cell culture technology has shown great promise as an alternative means of production for valuable natural products, particularly secondary metabolites that serve as agricultural chemicals, dyes, flavors, and medicinals.  However, low yields and variability in production put limits on the commercial use of this technology.  Plant cell culture research has recently shifted from a focus on “random” optimization of culture environmental conditions to a focus on developing an understanding of secondary metabolism so that rational enhancement strategies can be effectively applied.  The primary model system of use in our laboratory is the Taxus plant cell culture system for the production of the anti-cancer agent paclitaxel.  Paclitaxel is a member of a family of related compounds known as taxanes.  In Taxus suspension cultures, paclitaxel is often less than 10% of the total taxanes present.  This low selectivity represents an excellent opportunity to increase overall accumulation.  This talk will highlight our research-to-date, focusing on the application of molecular approaches to identify rate-limiting steps in paclitaxel accumulation for metabolic engineering targets.

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