Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginko biloba cells

作   者：张楠, 韩振泰,孙桂玲, Hoffman A, Wilson IW, 杨艳芳, 高茜, 吴建强, 谢丹, 戴均贵, 邱德有
期刊名称：Biochemical and Biophysical Research Communications
影响因子：2.831
卷 期 号：443(3)
页     码：938-943
关键词：Taxol, Taxoid 9a-hydroxylase, Ginkgo biloba, CYP450, Cell-free protein synthesis system LC朚S
论文摘要：
Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9a-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9a-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9a-hydroxylation activity. This work will aid future identification of the taxoid 9a-hydroxylase gene from Taxus sp.