Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from <it>Coleus forskohlii </it>Briq

• Main Authors: Kawamukai Makoto, Taura Futoshi, Engprasert Surang, Shoyama Yukihiro
• Format: Article
• Language: English
• Published: BMC 2004-11-01
• Series: BMC Plant Biology
• Online Access: http://www.biomedcentral.com/1471-2229/4/18

<p>Abstract</p> <p>Background</p> <p>Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the <it>GGPP synthase </it>gene in <it>Coleus forskohlii </it>Briq.</p> <p>Results</p> <p>The open reading frame for full-length <it>GGPP synthase </it>encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of <it>C. forskohlii GGPP synthase </it>amino acid sequences revealed high homologies with other plant <it>GGPP synthases</it>. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of <it>C. forskohlii </it>GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in <it>Escherichia coli </it>harboring pACCAR25Δ<it>crtE </it>from <it>Erwinia uredovora </it>and plasmid carrying <it>C. forskohlii GGPP synthase</it>. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, <it>C. forskohlii GGPP synthase </it>expression was strong in leaves, decreased in stems and very little expression was observed in roots.</p> <p>Conclusion</p> <p>This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.</p>