title: Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.
abstract: 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 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-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 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, The Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Forest Ecological Environment of Ministry of Forestry, Research Institute of Forest Ecology Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China.
authors.forename: Zhentai
authors.initials: Z
authors.lastname: Han
authors.affiliation: Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China.
authors.forename: Guiling
authors.initials: G
authors.lastname: Sun
authors.affiliation: Department of Chemistry, University of Portland, Portland, OR 97203, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Hoffman
authors.affiliation: CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2001, Australia.
authors.forename: Iain W
authors.initials: IW
authors.lastname: Wilson
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, The Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Forest Ecological Environment of Ministry of Forestry, Research Institute of Forest Ecology Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
authors.forename: Jungui
authors.initials: J
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, The Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China. Electronic address: qiudy@caf.ac.cn.
authors.forename: Deyou
authors.initials: D
authors.lastname: Qiu
mesh_terms: D000085:Acetates / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020441:Ginkgo biloba / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein
chemical_list: D000085:Acetates; D004224:Diterpenes; C484850:sinenxan A; D003577:Cytochrome P-450 Enzyme System
keywords: CYP450; Cell-free protein synthesis system; Ginkgo biloba; LC–MS; Taxoid 9α-hydroxylase; Taxol