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

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• 作者：Steven Edgar ; Fu-Shuang Li ; Kangjian Qiao ; Jing-Ke Weng ; Gregory Stephanopoulos
• 刊名：ACS Synthetic Biology
• 出版年：2017
• 出版时间：February 17, 2017
• 年：2017
• 卷：6
• 期：2
• 页码：201-205
• 全文大小：309K
• ISSN：2161-5063

文摘

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.