Development of Genetic Tools for Engineering Secondary Metabolism in Taxus Plant Cell Culture

Newton, Cassandra

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Development of Genetic Tools for Engineering Secondary Metabolism in Taxus Plant Cell Culture

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Plant cell culture (PCC) biomanufacturing is rapidly becoming an effective strategy for production of high-value plant natural products, such as therapeutic small molecules, vaccine adjuvants, and nutraceuticals. Yet, improving yields in non-model PCC systems is challenging due to limited understanding of their complex native biology and lack of metabolic engineering tools. In this thesis, I develop three unique approaches for understanding and manipulating metabolism in Taxus chinensis PCC, which is the primary industrial-scale platform host used for production of the chemotherapeutic drug paclitaxel (Taxol®). First, I pioneered an approach to mitigate epigenetic downregulation of metabolism in continuously subcultured plant cell lines using 5-azacytidine (5AC), a DNA methyltransferase inhibitor. Through treatment of 26-year-old cell lines with a combination of 5AC and the elicitor methyl jasmonate, we recovered paclitaxel production and expression of taxane biosynthetic pathway genes to levels comparable to recently initiated cell lines. Second, I used both chemical inhibitors and CRISPR-guided DNA methylation to control flux through the phenylpropanoid biosynthetic pathway, which contributes a phenylalanine derivative to the biosynthesis of paclitaxel and thus competes for precursors with taxane biosynthesis. Through targeted knockdown of PAL (the first committed step in phenylpropanoid biosynthesis) using a CRISPR-guided plant DNA methyltransferase, we achieved over a 25-fold increase in paclitaxel accumulation. Finally, I identified rate-limiting steps in paclitaxel biosynthesis through constitutive overexpression of four taxane biosynthetic pathway genes: TASY, DBAT, BAPT, and DBTNBT. Overexpression of each of these genes resulted in up to an 8-fold increase in paclitaxel yield and concerted activation of nearly the entire taxane biosynthetic pathway and associated transcription factors. This revealed that many taxane biosynthetic pathway genes are tightly co-regulated and confirmed that DBAT serves as a highly regulated rate-controlling step for paclitaxel biosynthesis. Ultimately, this work improves our knowledge of the intricate biology unique to medicinal plants and pioneers novel metabolic engineering tools for rewiring PCCs into next-generation chassis for production of societally valuable compounds.

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