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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

Year Degree Awarded

2015

Month Degree Awarded

September

First Advisor

Susan C. Roberts

Subject Categories

Biochemical and Biomolecular Engineering

Abstract

Plants produce a diversity of natural products that have commercial applications as flavorings, fragrances, pesticides and pharmaceuticals. These compounds are often the result of specialized metabolic pathways that are unique to plant systems, and have complex structures that make chemical synthesis routes infeasible. This necessitates exploitation of biological production routes. This thesis work presents a multi-scale characterization and engineering approach to understand and manipulate plant cell cultures on the extracellular (culture) and intracellular (metabolic pathway) levels. Studies focus on the commercially relevant suspension culture system Taxus, a medicinal plant species used for production of the FDA-approved anticancer drug paclitaxel.

Extracellular engineering: One of the unique characteristics of plant cell cultures is the tendency to grow in aggregates, which range in size from 50 to 2,000 µm in diameter. In Taxus, smaller aggregates accumulate higher levels of paclitaxel. Studies have looked at the effect of aggregation on product accumulation, but no methods have been developed to control aggregation properties in culture. In this study, a method was developed to decrease the mean aggregate size of Taxus suspension cultures through applied mechanical shear. Long-term application of shear did not affect culture growth and the mean aggregate size of the sheared population was reduced when compared to an unsheared control. Despite these promising results, the mean aggregate size of the sheared population fluctuated by over two-fold throughout the course of the experiment, indicating a lack of control over the aggregation dynamics. As a result, a population balance equation model was developed, which was used to determine the amount of mechanical shear necessary to reach a target aggregate size distribution, providing a new approach for controlling culture-level properties.

Intracellular engineering: In addition to conserved metabolic pathways, which are pathways involved in growth and development, plants have specialized metabolic pathways that allow them to adapt to their environment. These specialized pathways are often species specific and, as a result, are poorly defined. Additionally, the interactions between conserved and specialized metabolism are poorly understood, limiting the application of metabolic engineering strategies. To better understand global specialized metabolism, methods were developed to characterize active pathways in Taxus. It was found that all Taxus cultures divert carbon flux towards the production of phenolics, flavonoids and lignin, along with paclitaxel and related taxanes. Additionally, production of these compounds varies significantly over time and is directly related to culture aggregate size. These studies are amongst the first to address global specialized metabolism (consisting of both cooperative and competing pathways) in a non-model plant species and provide valuable insights into the design of effective metabolic engineering strategies to promote production of a particular class of products.

Due to the high level of interaction amongst cells in these complex cellular systems, successful engineering efforts must look beyond the level of the metabolic pathway. This thesis characterized and manipulated Taxus cultures on multiple scales to allow for the effective engineering of cultures for increased paclitaxel production.

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