Pan Xichun, Chen Min, Liu Yan, Wang Qiang, Zeng Lingjiang, Li Lianqiang, Liao Zhihua
Lab of Natural Products and Metabolic Engineering, Chongqing Sweetpotato Research Center, Key Lab of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, People's Republic of China.
DNA Seq. 2008 Apr;19(2):98-105. doi: 10.1080/10425170701446509.
Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. Here we report cloning, characterization and functional expression of a new cDNA encoding IPI from Camptotheca acuminata. The full-length cDNA was 1143 bp long designated as CaIPI (GenBank Accession Number: DQ839416), containing an open reading frame (ORF) of 930bp which encodes a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CaIPI was highly homologous with other IPI gene and the deduced amino acid sequence of CaIPI was similar to known plant IPIs and contained Cys-149 and Glu-212 active sites. Phylogenic analysis indicated that all IPIs could be divided into five groups and CaIPI belonged to plant IPIs' family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues. The result showed that CaIPI expression could be detected in roots, stems and tender leaves but could not in mature leaves and fruits, and the expression levels was much higher in stems than in roots and tender leaves. Finally, CaIPI was functionally expressed in engineered Escherichia coli in which the carotenoid pathway was reconstructed. In engineered E. coli, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange beta-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil the biosynthetic mechanism of camptothecin precursors.
异戊烯基二磷酸异构酶（EC 5.3.3.2，IPI）催化5碳异戊烯基二磷酸（IPP）及其烯丙基异构体二甲基烯丙基二磷酸（DMAPP）的可逆转化，这两种物质是类异戊二烯（包括抗肿瘤药物喜树碱）的必需前体。在此，我们报道了从喜树中克隆、鉴定并功能性表达一个编码IPI的新cDNA。该全长cDNA长1143 bp，命名为CaIPI（GenBank登录号：DQ839416），包含一个930 bp的开放阅读框（ORF），编码一个309个氨基酸的多肽。生物信息学分析表明，CaIPI的cDNA序列与其他IPI基因高度同源，推导的CaIPI氨基酸序列与已知植物IPIs相似，包含Cys-149和Glu-212活性位点。系统发育分析表明，所有IPIs可分为五组，CaIPI属于植物IPIs家族。进行了组织表达谱分析以研究CaIPI在不同组织中的转录水平。结果表明，在根、茎和嫩叶中可检测到CaIPI表达，但在成熟叶和果实中未检测到，且茎中的表达水平远高于根和嫩叶。最后，CaIPI在重建了类胡萝卜素途径的工程大肠杆菌中实现了功能性表达。在工程大肠杆菌中，CaIPI可促进代谢流进入类胡萝卜素生物合成，使细菌产生橙色的β-胡萝卜素。这些证实了CaIPI具有IPI基因的典型功能。总之，CaIPI的克隆、鉴定和功能性表达将有助于在分子遗传学水平上理解CaIPI的功能，并揭示喜树碱前体的生物合成机制。
