Mechanistic insights and engineering of Paclitaxel biosynthetic pathway

The plant natural product Paclitaxel (Taxol®), stands as one of the most medically and economically significant natural products in use today.  However, despite its importance, this compound is currently only obtained at low concentrations from harvested plant tissue, or non-recombinant plant cell culture, as attempts at microbial production has been met with limited success.  Here, we report on mechanistic studies and engineering efforts to identify and overcome key barriers in the Taxol biosynthetic pathway.  We identify a previously uncharacterized bottleneck, arising from the wide product distribution of the first enzymatic step, catalyzed by a cytochrome P450.  We then probe the drivers of this poor selectivity both chemically and on an enzymatic level and propose an alternative enzymatic mechanism.  This mechanistic insight is then leveraged to improve pathway yield and selectivity by proposing and validating three distinct routes to improvement and evaluating each one independently.  This work is not lays the foundation for fully elucidating the Taxol biosynthetic pathway but is also essential for reliable, scalable production of Taxol.