A new isopentenyl diphosphate isomerase gene from Camptotheca acuminata: cloning, characterization and functional expression in Escherichia coli.
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.
Pan
Xichun
X
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.
Chen
Min
M
Liu
Yan
Y
Wang
Qiang
Q
Zeng
Lingjiang
L
Li
Lianqiang
L
Liao
Zhihua
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D029769:Camptotheca / Q000201:enzymology* / Q000235:genetics*; D002166:Camptothecin / Q000096:biosynthesis* / Q000737:chemistry; D019748:Carbon-Carbon Double Bond Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005816:Genetic Complementation Test; D045782:Hemiterpenes; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity / Q000235:genetics
D045782:Hemiterpenes; D007527:Isoenzymes; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D002166:Camptothecin