title: Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate.
abstract: 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.
authors: Steven
authors: S
authors: Edgar
authors: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
authors: Fu-Shuang
authors: FS
authors: Li
authors: Kangjian
authors: K
authors: Qiao
authors: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
authors: Jing-Ke
authors: JK
authors: Weng
authors: Gregory
authors: G
authors: Stephanopoulos
mesh_terms: D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006900:Hydroxylation / Q000235:genetics; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism*; D010084:Oxidation-Reduction; D017239:Paclitaxel / Q000378:metabolism*
chemical_list: D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel