Li, C.; Sun, X.; Chen, R.; Xie, K.; Chen, D.; Liu, J.; Dai, J.

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. Here we functionally characterized two novel taxane C13-O-deacetylases (T13dA1 and T13dA2) from Taxus media cell cultures, providing experimental evidence for the molecular and biochemical plausibility of C13-O-deacetylation in paclitaxel biosynthesis in Taxus species. Also, we identified a previously uncharacterized bifunctional taxane C7{beta}-O-, C9-O-deacetylase, designated T79dA, which demonstrates the functional promiscuity by enabling stepwise deacetylation at taxane C7{beta} and C9 positions in a single enzymatic reaction. Furthermore, T7dA1, a novel taxane C7{beta}-O-deacetylase with higher activity than the reported T7dA was discovered and characterized here. Moreover, we reconstituted two new pathways (an 18-gene and a 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. These pathways with the previously established 17-gene baccatin III pathway, further allow paclitaxel biosynthesis to be a network. Our reconstituted 19-gene pathway achieves a baccatin III yield of up to 23 g g-1 dried weight (DW) in N. benthamiana leaves, which is comparable to the yield reported for the 17-gene pathway. This work facilitates a better understanding, elucidation and reconstruction of metabolic network of paclitaxel biosynthetic pathway, and provides new enzymes and strategies for artificial pathway reconstruction and efficiently bio-chemical production of paclitaxel.