Missing taxane C13α-O-deacetylases reveal a cryptic acetylation-deacetylation module in paclitaxel biosynthesis

The prevalence of naturally occurring C13-acetoxy taxanes, together with the presence of a native C13-acetyltransferase in yew trees, suggests that the natural biosynthetic pathway for paclitaxel may involve a cryptic C13-O-deacetylation step. However, whether a putative taxane C13-O-deacetylase (T13dA) acts in the pathway of paclitaxel biosynthesis remains elusive. Herein, we functionally characterized two novel taxane C13-O-deacetylases (T13dA1 and T13dA2) from Taxus x media cell cultures, providing experimental evidence for the molecular and biochemical plausibility of C13-O-deacetylation in paclitaxel biosynthesis in Taxus species. Furthermore, T7dA1, a novel taxane C7{beta}-O-deacetylase with higher activity than the reported T7dA was discovered and characterized here. Notably, we reconstituted a new 19-gene pathway enabled by the integration of a C13-O-acetylation-deacetylation module for the de novo biosynthesis of baccatin III in Nicotiana benthamiana leaves, reaching a yield of 71{square} g{square} g-1 DW, much higher than that obtained from the 17{square} gene pathway lacking this module. These findings not only demonstrate the functional importance of the C13-O-acetylation-deacetylation module as a branch within the paclitaxel biosynthetic network, but also expand the multi-branched taxane metabolic network in Taxus and provide novel enzymatic tools and engineering strategies to facilitate heterologous pathway reconstitution and efficient paclitaxel production.