Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library.
Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562 aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.
Bertea
Cinzia M
CM
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Voster
Alessandra
A
Verstappen
Francel W A
FW
Maffei
Massimo
M
Beekwilder
Jules
J
Bouwmeester
Harro J
HJ
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D031172:Artemisia annua / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D045788:Sesquiterpenes, Germacrane / Q000096:biosynthesis*; D013729:Terpenes / Q000378:metabolism
D045788:Sesquiterpenes, Germacrane; D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C511057:germacrene A synthase, Artemisia annua