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Amorpha-4,11-diene Synthase of Artemisia annua: cDNA Isolation and Bacterial Expression of a Terpene Synthase Involved in Artemisinin Biosynthesis☆

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Abstract

Artemisia annua, an indigenous plant to Korea, contains 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 the cyclization of farnesyl diphosphate to produce a sesquiterpene, which was identified through GC–MS analysis as amorpha-4,11-diene.

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    ☆

    The nucleotide sequence data reported in this article have been deposited with the GenBank/EMBL Database under Accession No. AJ251751.

    2

    The first two coauthors share equal credit in this investigation.

    3

    To whom correspondence and reprint requests should be addressed. Fax: 82-31-293-8608. E-mail: [email protected].

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