Engineering of 10‐Deacetylbaccatin III‐10‐β‐O‐Acetyltransferase From <i>Taxus</i> Species for Efficient Acetylating Non‐Natural Substrates Into Taxol in <i>Nicotiana benthamiana</i>

7-β-xylosyl-10-deacetyltaxol (XDT) is much more abundant than the anticancer drug Taxol in Taxus species and is usually regarded as the by-product of Taxol. It could be enzymatically transformed into 10-deacetyltaxol (DT), and the latter could be further converted into Taxol. The enzyme 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) can acetylate the non-natural substrate DT into Taxol, but the conversion efficiency was extremely low. Herein, we globally redesigned DBAT_cus from Taxus cuspidata to improve its efficiency in DT acetylation through combinatorial protein engineering strategies including virtual saturation mutagenesis, in silico screening, DNA shuffling, and iterative combinatorial mutagenesis. Several more active DBAT mutants against DT were obtained, among which the ICM9-6 exhibited 16.4 times higher activity than DBAT_cus. The transient expression system of Nicotiana benthamiana was then established, and the ICM9-6 was functionally expressed in the system, with yield of 8.2 μg g^-1 FW (129.3 μg g^-1 DW) Taxol when the system was fed with DT. Specifically, the fungal glycoside hydrolase LXYL-P1-2 that was responsible for converting XDT into DT was also functionally expressed in the system, and upon feeding XDT, the co-expression of LXYL-P1-2 and ICM9-6 yielded 3.6 μg g^-1 FW (55.4 μg g^-1 DW) Taxol. These results represent the highest reported Taxol productivity in the tobacco system to date and lay a foundation for the construction of the stable transgenic cell lines of tobacco and more efficiently converting DT or XDT into Taxol for the large-scale pharmaceutical manufacturing.