Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

C.M. Bertea, A. Voster, F.W.A. Verstappen, M. Maffei, M.J. Beekwilder, H.J. Bouwmeester

Research output: Contribution to journalArticleAcademicpeer-review

90 Citations (Scopus)

Abstract

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 562aa. 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.
Original languageEnglish
Pages (from-to)3-12
JournalArchives of Biochemistry and Biophysics
Volume448
Issue number1-2
DOIs
Publication statusPublished - 2006

Fingerprint

Artemisia annua
Trichomes
Cloning
Biosynthesis
Terpenes
Gene Library
Organism Cloning
Expressed Sequence Tags
Mevalonic Acid
Asteraceae
Sesquiterpenes
Escherichia coli Proteins
Cyclization
Biosynthetic Pathways
Antimalarials
Recombinant Proteins
Escherichia coli
Open Reading Frames
Plasmids
Complementary DNA

Keywords

  • amorpha-4,11-diene synthase
  • bacterial expression
  • arabidopsis-thaliana
  • diphosphate synthase
  • molecular-cloning
  • key enzyme
  • l.
  • (e)-beta-farnesene
  • sesquiterpenes
  • accumulation

Cite this

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title = "Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library",
abstract = "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 562aa. 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.",
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author = "C.M. Bertea and A. Voster and F.W.A. Verstappen and M. Maffei and M.J. Beekwilder and H.J. Bouwmeester",
year = "2006",
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language = "English",
volume = "448",
pages = "3--12",
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}

Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. / Bertea, C.M.; Voster, A.; Verstappen, F.W.A.; Maffei, M.; Beekwilder, M.J.; Bouwmeester, H.J.

In: Archives of Biochemistry and Biophysics, Vol. 448, No. 1-2, 2006, p. 3-12.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

AU - Bertea, C.M.

AU - Voster, A.

AU - Verstappen, F.W.A.

AU - Maffei, M.

AU - Beekwilder, M.J.

AU - Bouwmeester, H.J.

PY - 2006

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N2 - 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 562aa. 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.

AB - 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 562aa. 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.

KW - amorpha-4,11-diene synthase

KW - bacterial expression

KW - arabidopsis-thaliana

KW - diphosphate synthase

KW - molecular-cloning

KW - key enzyme

KW - l.

KW - (e)-beta-farnesene

KW - sesquiterpenes

KW - accumulation

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DO - 10.1016/j.abb.2006.02.026

M3 - Article

VL - 448

SP - 3

EP - 12

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

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ER -