Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate.

Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5α-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors.