Metabolic engineering of Taxus chinensis suspension cell lines for overproduction of paclitaxel reveals insights into pathway regulation.

Plant cell culture is a growing platform for sustainable biomanufacturing of plant-derived compounds, including the chemotherapeutic paclitaxel, which is produced industrially using Taxus chinensis plant cell culture. While strategies for improving yields of natural products in plant cell culture through elicitation of secondary metabolism have been widely studied, genetic approaches for manipulating metabolism have remained underexplored. Here, we constitutively overexpress four different genes in the paclitaxel biosynthetic pathway in T. chinensis plant cell culture and elucidate effects on pathway regulation and taxane biosynthesis. Overexpression of each of these four genes - taxadiene synthase (TASY), 10-deacetylbaccatin III-10-O-transferase (DBAT), baccatin-aminophenylpropanoyl-13-O-transferase (BAPT), and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT) - resulted in a 2-8-fold increase in paclitaxel accumulation, with overexpression of DBAT resulting in the largest increase. We also identified DBAT as a key rate-controlling step in biosynthesis of not only paclitaxel, but other taxane-derived compounds, as its overexpression resulted in significantly increased accumulation of taxane impurities. Expression profiling of all four transgenic cell lines revealed concerted activation of taxane metabolism, with upregulation of nearly all pathway genes and transcription factors. DBAT was the notable exception to this concerted activation and was only upregulated in the cell line specifically targeting that gene, indicating that DBAT is likely regulated differently and much more tightly than all other genes in the pathway. While this work specifically developed tools for engineering Taxus plant cell cultures, more broadly, these results illustrate the importance of genetic engineering of plant cell systems to achieve optimum yields. Through the application of modern synthetic biology and metabolic engineering tools to plant cell culture, we can enable rapid cell line optimization for production of specialized metabolites.