Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq.
BACKGROUND
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 GGPP synthase gene in Coleus forskohlii Briq.


RESULTS
The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25DeltacrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.


CONCLUSIONS
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.
Engprasert
Surang
S
Graduate School of Pharmaceutical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. engprasert@yahoo.com <engprasert@yahoo.com>
Taura
Futoshi
F
Kawamukai
Makoto
M
Shoyama
Yukihiro
Y
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D031330:Coleus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005576:Colforsin / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005578:Fosfomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018547:Plant Stems / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000166:cytology; D014162:Transfection
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005576:Colforsin; D005578:Fosfomycin; C024640:fosmidomycin; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase