Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis

  • Chang Y
  • Song S
  • Park S
 et al. 
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Artemisia annua, an indigenous plant to Korea, conrains an antimalarial sesquiterpene, artemisinin. The first committed step of artemisinin biosynthesis is the cyclization of farnesyl diphosphate by a sesquiterpene synthase to produce an amorphane-type ring system. The aims of this research were to molecularly clone and express amorpha-4,11-diene synthase for metabolic engineering. PCR amplification of genomic DNA with a pair of primers, designed from the conserved regions of sesquiterpene synthases of several plants, produced a 184-bp DNA fragment. This fragment was used in Northern blot analysis as a probe, showing approximately 2.2 kb of a single band. Its Sequence information was used to produce 2106 bp of a full-length cDNA sequence including 1641 bp of open reading frame for 546 amino acids (kcs12) through a rapid amplification of cDNA ends (RACE). The deduced amino acid sequence displayed 36% identity with 5-epi-aristolochene synthase of Nicotiana tabacum. A soluble fraction of Escherichia coli harboring kcs12 catalyzed tile cyclization of farnesyl diphosphate to produce a sesquiterpene, which was identified through GC-MS analysis as amorpha-4,11-diene. (C) 2000 Academic Press.

Author-supplied keywords

  • Amorpha-4,11-diene synthase
  • Artemisia annua
  • Artemisinin biosynthesis
  • Rapid amplification of cDNA ends
  • Sesquiterpene synthase

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