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青蒿紫穗槐-4,11-二烯P450氧化酶及还原酶基因的克隆及功能鉴定

发表时间:2011-06-15  浏览量:1266  下载量:371
全部作者: 孔建强,郑海娜,支晓慧,史彦薇,王伟,朱平,刘振娅
作者单位: 中国医学科学院 & 北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室
摘 要: 为构建能规模化制备青蒿酸的微生物工程菌,需要对青蒿酸生物合成途径中的酶基因进行功能鉴定。提取青蒿植物RNA,通过RT-PCR克隆得到2个青蒿酸生物合成途径相关酶基因:紫穗槐-4,11-二烯P450氧化酶(cytochrome P450 monooxygenase,CYP71AV1)及其电子配偶体P450还原酶(NADPH:cytochrome P450 oxidoreductase,CPR)基因,将这2个基因以及青蒿紫穗槐-4,11-二烯合酶基因、酵母的HMG-CoA还原酶(3-hydroxy-3-methylglutaryl-coenzymeA reductase,HMGR)基因、FPP合酶(farnesyl diphosphate synthase,FPPS)基因导入酿酒酵母WK1,构建能合成青蒿酸的酵母代谢工程菌。通过高效液相色谱法(high performance liquid chromatography,HPLC)和傅里叶回旋共振质谱,在青蒿酸酵母工程菌中检测到了青蒿酸,表明克隆得到的P450氧化酶及P450还原酶基因具备功能,从而为通过合成生物学技术制备青蒿素前体奠定了基础。
关 键 词: 微生物药物学;P450氧化酶;P450还原酶;青蒿酸;酵母工程菌
Title: cDNA cloning and function characterization of Artemisia annua L. genes encoding cytochrome P450 monooxygenase and P450 oxidoreductase
Author: KONG Jianqiang, ZHENG Haina, ZHI Xiaohui, SHI Yanwei, WANG Wei, ZHU Ping, LIU Zhenya
Organization: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College
Abstract: The total RNA of Artemisia annua L. plant was extracted and transcripted into cDNA. Two genes encoding amorpha-4,11-diene cytochrome P450 monooxygenase (CYP71AV1) and its redox partner NADPH: cytochrome P450 oxidoreductase (CPR) were cloned from the Artemisia annua L. cDNA. All cloned cDNAs were confirmed by DNA sequencing. A Saccharomyces cerevisiae expression vector pYG450Gre containing CYP71AV1 and CPR genes was constructed. pYG450Gre, pGBT9/A/HMG/G/FPP and pGADADS were co-transformed into S.cerevisiae WK1 to form the engineered yeasts producing artemisinic acid by LiOAc-mediated method. The identification of positive clone and the fermentation of the transformants were performed. The fermentation metabolites were also extracted and analyzed by high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry. The result showed the identification of artemisinic acid from engineered yeast compared with the artemisinic acid standard, which indicated the CYP71AV1 and CPR genes function in engineered S. cerevisiae. These results were not yet reported in China.
Key words: microbial medicine; cytochrome P450 monooxygenase; NADPH: cytochrome P450 oxidoreductase; artemisinic acid; engineered Saccharomyces cerevisiae
发表期数: 2011年6月第11期
引用格式: 孔建强,郑海娜,支晓慧,等. 青蒿紫穗槐-4,11-二烯P450氧化酶及还原酶基因的克隆及功能鉴定[J]. 中国科技论文在线精品论文,2011,4(11):967-974.
 
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