title: Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis.
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.
authors.affiliation: School of Agricultural Biotechnology and the Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon, Korea.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chang
authors.forename: S H
authors.initials: SH
authors.lastname: Song
authors.forename: S H
authors.initials: SH
authors.lastname: Park
authors.forename: S U
authors.initials: SU
authors.lastname: Kim
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D019306:Artemisia / Q000201:enzymology*; D037621:Artemisinins; D015152:Blotting, Northern; D015139:Blotting, Southern; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes / Q000737:chemistry*; D014026:Nicotiana / Q000201:enzymology
chemical_list: D037621:Artemisinins; D018076:DNA, Complementary; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; C031327:artemisinin; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase