Metabolic Engineering of Taxane Biosynthesis in Taxus chinensis Plant Cell Culture

Gandhi, Jay; Harrison, Alexandra; Horton, Sean; Revene, Lauren

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Metabolic Engineering of Taxane Biosynthesis in Taxus chinensis Plant Cell Culture

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Ovarian, lung, and breast cancer impact millions globally and are a common cause of death in the United States. These cancerous diseases can be treated with the chemotherapeutic paclitaxel (Taxol), which is naturally produced by the Taxus plant genus. While there is a demonstrated need for increased paclitaxel production, many production methods are unsustainable or result in low yield. Since plant cell culture (PCC) has been found to be a costly process with slow plant growth and low yield, a thorough understanding of the paclitaxel biosynthetic pathway would be beneficial to efficiently increase yield. Metabolic engineering of the paclitaxel biosynthetic pathway could be used to elucidate rate-limiting steps in the pathway and offer more specific and highly tunable control of metabolism. To this end, the primary objective for this research project is to develop tools to enable metabolic engineering of Taxus chinensis PCC and determine rate-limiting steps in the paclitaxel biosynthetic pathway through overexpression of four genes identified as potentially rate-limiting steps: BAPT, DBAT, DBTNBT, and TASY. First, we developed a stable Agrobacterium infiltration-based transformation method for Taxus chinensis cell line 48.82A.3s and identified a suitable selectable marker that effectively killed non-transgenic cells (10 μg/mL of hygromycin). Transgenic Taxus chinensis cell lines overexpressing each of the four taxane biosynthetic pathway genes were generated using this method and a large (several hundred-fold) increase in gene expression was confirmed using RT-qPCR. Taxane production measured using UPLC, however, did not have a statistically significant increase. Additionally, vectors for overexpression of the studied genes fused with a C-terminal mCherry tag were successfully constructed and transformed into A. tumefaciens EH105, but a successful transformation into Taxus chinensis was not completed due to time constraints and contamination issues. Homology modeling of mCherry fusion proteins show that mCherry should not interfere with the active site of the fused enzyme.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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