title: Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua.
abstract: Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Arman Beyraghdar
authors.initials: AB
authors.lastname: Kashkooli
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud, 4-5, Box L7.07.14, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Sallets
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Hieng-Ming
authors.initials: HM
authors.lastname: Ting
authors.affiliation: Laboratory of Cell Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Norbert C A
authors.initials: NCA
authors.lastname: de Ruijter
authors.affiliation: Department of Chemistry and Biomedical Sciences, Linnaeus University, SE-38192 Kalmar, Sweden.
authors.forename: Linda
authors.initials: L
authors.lastname: Olofsson
authors.affiliation: Department of Chemistry and Biomedical Sciences, Linnaeus University, SE-38192 Kalmar, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Brodelius
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud, 4-5, Box L7.07.14, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Pottier
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud, 4-5, Box L7.07.14, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Harro
authors.initials: H
authors.lastname: Bouwmeester
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: van der Krol
mesh_terms: D031172:Artemisia annua / Q000502:physiology*; D037621:Artemisinins / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000502:physiology*
chemical_list: D037621:Artemisinins; D002352:Carrier Proteins; D010940:Plant Proteins; C030363:lipid transfer protein; C031327:artemisinin
keywords: ABC transporters; Artemisia annua; Artemisinin; Lipid transfer proteins; Nicotiana benthamiana; Pleiotropic Drug Resistance protein