title: Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism.
abstract: Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using13C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Czechowski
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: Tony R
authors.initials: TR
authors.lastname: Larson
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Catania
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Harvey
authors.affiliation: Department of Chemistry, University of Reading, Reading RG6 6AD, United Kingdom.
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Brown
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom; ian.graham@york.ac.uk.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Graham
mesh_terms: D000962:Antimalarials / Q000378:metabolism*; D031172:Artemisia annua / Q000235:genetics* / Q000378:metabolism; D037621:Artemisinins / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D003433:Crosses, Genetic; D018744:DNA, Plant / Q000235:genetics; D018628:Gene Dosage; D005838:Genotype; D016296:Mutagenesis; D009154:Mutation; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics; D000081223:Polycyclic Sesquiterpenes; D020641:Polymorphism, Single Nucleotide; D012717:Sesquiterpenes / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism; D064201:Trichomes
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D018744:DNA, Plant; D010940:Plant Proteins; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; D013729:Terpenes; C515348:amorpha-4,11-diene; C520462:dihydroartemisinic acid; C031327:artemisinin
keywords: Artemisia annua; artemisinin; p450 oxidase; sesquiterpene; terpenoid