title: The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage.
abstract: Artemisia annua, which produces the anti-malaria compound artemisinin, occurs as high-artemisinin production (HAP) and low-artemisinin production (LAP) chemotypes. Understanding the basis of the difference between these chemotypes would assist breeding and optimising artemisinin biosynthesis. Here we present a systematic comparison of artemisinin biosynthesis genes that may be involved in determining the chemotype (CYP71AV1, DBR2 and ALDH1). These genes were isolated from the two chemotypes and characterized using transient expression in planta. The enzyme activity of DBR2 and ALDH1 from the two chemotypes did not differ, but structural differences in CYP71AV1 from LAP and HAP chemotypes (AMOLAP and AMOHAP, respectively) resulted in altered enzyme activity. AMOLAP displays a seven amino acids N-terminal extension compared with AMOHAP. The GFP fusion of both proteins show equal localization to the ER but AMOHAP may have reduced stability. Upon transient expression in Nicotiana benthamiana, AMOLAP displayed a higher enzyme activity than AMOHAP. However, expression in combination with the other pathway genes also resulted in a qualitatively different product profile ('chemotype'); that is, in a shift in the ratio between the unsaturated and saturated (dihydro) branch of the pathway.
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Hieng-Ming
authors: HM
authors: Ting
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Bo
authors: B
authors: Wang
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.|Plant Research International, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Anna-Margareta
authors: AM
authors: Rydén
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Lotte
authors: L
authors: Woittiez
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Teun
authors: T
authors: van Herpen
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Francel W A
authors: FWA
authors: Verstappen
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Carolien
authors: C
authors: Ruyter-Spira
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.|Plant Research International, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Jules
authors: J
authors: Beekwilder
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Harro J
authors: HJ
authors: Bouwmeester
authors: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
authors: Alexander
authors: A
authors: van der Krol
mesh_terms: D060054:Agrobacterium / Q000378:metabolism; D000595:Amino Acid Sequence; D037621:Artemisinins / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D005978:Glutathione / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
chemical_list: D037621:Artemisinins; D010940:Plant Proteins; C520462:dihydroartemisinic acid; C047721:artemisic acid; C031327:artemisinin; D005978:Glutathione
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