(640h) Quantifying Secondary Metabolic Pathway Flux and Time-Dependent Variation in Plant Suspension Cultures

Authors: 
Wilson, S. A., University Of Massachusetts Amherst
Roberts, S. C., University of Massachusetts, Amherst

Plants produce thousands of secondary metabolites that have high commercial value as pesticides, fragrances, flavors and pharmaceuticals. Due to the complex structures of these compounds, chemical synthesis routes are often not commercially feasible, necessitating the use of biological production platforms. Metabolic engineering can be used to increase the accumulation of products in these systems, but the complexity of the interaction between plant primary and secondary metabolism hinders these efforts. Additionally, plant secondary metabolism is species-specific, so pathways to high value secondary metabolites are often under-characterized. As a result of this complex metabolism, the effects of single gene manipulations on product accumulation are not easily predicted. To better understand the interactions between plant primary and secondary metabolism, methods were established to map the flux through key secondary pathways present in Taxus suspension cultures, which uniquely accumulate the anticancer compound paclitaxel. A transcriptomics study identified active pathways in cultures with induced secondary metabolism. Based on these data,  accumulation of broad classes of compounds (phenolics and flavonoids) and key endpoint products (Taxol™ and lignin) were quantified over time to assess variability in secondary metabolic flux.

The effect of an abiotic elicitor of secondary metabolism, methyl jasmonate (MJ), was determined over multiple generations of growth in both a paclitaxel accumulating and non-paclitaxel accumulating Taxus cell line. Key results included: 1) unelicited cultures exhibited variability in culture aggregation and base-line secondary metabolite levels; 2) inhibition of growth upon the addition of MJ did not consistently correlate to increased secondary metabolite production in both paclitaxel accumulating and non-accumulating cultures; 3) all cultures respond to MJ by increasing the production of lignin, a phenylpropanoid that diverts flux away from paclitaxel; and 4) all cultures accumulated detectable levels of phenolics and flavonoids, indicating that these pathways are active but not always induced by addition of MJ. These studies are among the first to utilize high throughput assays to investigate flux through key secondary metabolic pathways in plant metabolism and can serve as the basis for the study of other non-model plant systems. Specific results will enable the design of metabolic engineering strategies to increase Taxol™ accumulation in Taxus suspension cultures.