Should I stay or should I go? The fight or flight defense responses in Taxus cell culture

McKee, Michelle C

Etd

Should I stay or should I go? The fight or flight defense responses in Taxus cell culture

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Taxus culture is the current system to commercially produce paclitaxel, a highly potent and valuable chemotherapeutic drug, although paclitaxel biosynthesis and other cellular processes within Taxus remain largely elusive. The majority of the last 30 years in Taxus research has focused exclusively on paclitaxel biosynthesis without regard to concurrent and often antagonistic processes. This dissertation investigates parallel cellular events with respect to how they impact paclitaxel accumulation. Methyl jasmonate, a well-known elicitor to increase paclitaxel accumulation, also boosts the competing phenylpropanoid biosynthetic pathway and, at high concentrations, induces programmed cell death (PCD). An immediate method to detect PCD in Taxus culture was developed and applied to identify the threshold between paclitaxel biosynthesis and PCD. Next, Taxus cultures with different paclitaxel production capabilities were used to investigate the relationship between culture growth and the taxane and phenylpropanoid biosynthetic pathways. Caffeic acid was shown to inhibit taxane biosynthesis without interrupting culture growth or phenylpropanoid biosynthesis. Finally, paclitaxel accumulation is hypothesized to be limited by intracellular storage as product removal increases yields. Two distinct approaches, subcellular fractionation and immunohistochemistry, were used to confirm the plastids as the intracellular paclitaxel storage site rather than the vacuole. These results provide alternative opportunities to globally engineer Taxus cell culture instead of directly manipulating the biosynthetic pathway to increase paclitaxel yields. This unique systems approach can be applied to other plant cell cultures to comprehensively understand metabolism and elicitation and to boost metabolite production.

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