(6em) Multi-Level Engineering Approaches for Manipulating Plant Metabolism in Culture

Plants are a source of a vast diversity of natural products that have commercial applications as flavors, fragrances, pesticides and pharmaceuticals. These products are often the result of sophisticated secondary metabolic pathways that are specialized to plant cells. Due to the complex structures of these compounds, chemical synthesis is often infeasible, necessitating the use of biological production platforms. In this project, methods were developed and employed to characterize and engineer plant cell systems at both the environmental and molecular levels to decipher mechanisms for promoting plant secondary metabolism. Studies utilized suspension culture of the non-model plant system, Taxus. These cultures are utilized for commercial production of the anti-cancer agent paclitaxel (Taxol^TM, Bristol-Myers Squibb), which is used in the treatment of breast, lung and ovarian cancers, as well as AIDS-related Kaposi’s sarcoma.

Environmental manipulation: When plant cells divide, the cell walls do not fully separate, leading to the formation of aggregates, which can range in size from two to thousands of cells. These aggregates result in the formation of subpopulations within a culture. This heterogeneity is believed to be a major source of the inherent variability associated with plant production systems. To aid in the understanding of culture aggregation, a method was developed to reduce aggregation through the application of mechanical shear. A population balance model was established to determine the amount of shear required to reach a target aggregate size distribution. By applying shear over multiple generations of cell growth, a more heterogeneous culture with a constant mean aggregate size was developed with no affect on cell health.

Molecular-level manipulation: Although plant primary metabolism is well characterized and highly conserved across plant species, plant secondary metabolism is often species specific and, as a result, poorly defined. Additionally, the interface between primary and secondary metabolism is poorly understood, limiting metabolic engineering efforts. High-throughput methods were developed to evaluate the flux distribution through specific pathways involved in Taxus metabolism. It was found that cultures that produce paclitaxel also divert carbon flux towards the production of phenolics and flavonoids. A long-term study revealed high levels of variability in culture aggregation and secondary metabolite accumulation. These studies are amongst the first to study global secondary metabolism in a non-model plant species and provide valuable insight into the design of effective metabolic engineering strategies to promote production of a particular class of secondary products.

Due to the high level of interaction between cells in these complex cellular systems, successful engineering efforts must look beyond the level of the metabolic pathway. This project is the first of its kind to characterize and manipulate Taxus cultures on a multi-scale level to allow for the effective engineering of cultures for increased paclitaxel production. Moving forward, manipulation of culture aggregation through mechanical shearing and cellular metabolism through metabolic engineering could lead to more homogeneous cultures with reduced variability and increased paclitaxel accumulation.