The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage
Ting, H.M. ; Wang, B. ; Ryden, A.M. ; Woittiez, L.S. ; Verstappen, F.W.A. ; Ruyter-Spira, C.P. ; Beekwilder, M.J. ; Bouwmeester, H.J. ; Krol, A.R. van der; Herpen, T.W.J.M. van - \ 2013
New Phytologist 199 (2013)2. - ISSN 0028-646X - p. 352 - 366.
molecular-cloning - annua l - amorpha-4,11-diene synthase - dihydroartemisinic acid - mass-spectrometry - key role - arabidopsis - identification - metabolomics - precursors
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.
Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.)
Majdi, M. ; Liu, Q. ; Karimzadeh, G. ; Malboobi, M.A. ; Beekwilder, J. ; Cankar, K. ; Vos, C.H. de; Todorovic, S. ; Simonovic, A. ; Bouwmeester, H.J. - \ 2011
Phytochemistry 72 (2011)14-15. - ISSN 0031-9422 - p. 1739 - 1750.
germacrene-a synthase - artemisia-annua l - sesquiterpene lactones - gene-expression - amorpha-4,11-diene synthase - developmental regulation - migraine prophylaxis - double-blind - in-vitro - asteraceae
Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-ß-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur.
The molecular cloning of dihydroartemisinic aldehyde reductase and its implication in artemisinin biosynthesis in Artemisia annua
Ryden, A.M. ; Ruyter-Spira, C.P. ; Quax, W.J. ; Hiroyuki, O. ; Toshiya, M. ; Kayser, O. ; Bouwmeester, H.J. - \ 2010
Planta Medica 76 (2010). - ISSN 0032-0943 - p. 1778 - 1783.
chain dehydrogenases/reductases sdrs - amorpha-4,11-diene synthase - key enzyme - functional assignments - expression - acid - identification - plants - dehydrogenase/reductase - terpenoids
A key point in the biosynthesis of the antimalarial drug artemisinin is the formation of dihydroartemisinic aldehyde which represents the key difference between chemotype specific pathways. This key intermediate is the substrate for several competing enzymes, some of which increase the metabolic flux towards artemisinin, and some of which - as we show in the present study - may have a negative impact on artemisinin production. In an effort to understand the biosynthetic network of artemisinin biosynthesis, extracts of A. annua flowers were investigated and found to contain an enzyme activity competing in a negative sense with artemisinin biosynthesis. The enzyme Red1 is a broad substrate oxidoreductase belonging to the short chain dehydrogenase/reductase family with high affinity for dihydroartemisinic aldehyde and valuable monoterpenoids. Spatial and temporal analysis of cDNA revealed Red1 to be trichome specific. The relevance of Red1 to artemisinin biosynthesis is discussed.
Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.
Ryden, A.M. ; Ruyter-Spira, C.P. ; Litjens, R. ; Takahashi, S. ; Quax, W.J. ; Osada, H. ; Bouwmeester, H.J. ; Kayser, O. - \ 2010
Plant and Cell Physiology 51 (2010)7. - ISSN 0032-0781 - p. 1219 - 1228.
chain dehydrogenases/reductases sdrs - amorpha-4,11-diene synthase - functional assignments - biosynthetic-pathway - essential oil - key enzyme - expression - reductase - peppermint - acid
From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, and in particular functions as a menthone:neomenthol oxidoreductase. The kinetic parameter kcat/Km was determined to be 939 fold more efficient for the reduction of (-)-menthone to (+)-neomenthol, than results previously reported for the menthone:neomenthol reductase from Mentha x piperita. Based on its kinetic properties, the possible use of Red1 in biological crop protection is discussed.
Isoprenoid biosynthesis in Artemisia annua: Cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library
Bertea, C.M. ; Voster, A. ; Verstappen, F.W.A. ; Maffei, M. ; Beekwilder, M.J. ; Bouwmeester, H.J. - \ 2006
Archives of Biochemistry and Biophysics 448 (2006)1-2. - ISSN 0003-9861 - p. 3 - 12.
amorpha-4,11-diene synthase - bacterial expression - arabidopsis-thaliana - diphosphate synthase - molecular-cloning - key enzyme - l. - (e)-beta-farnesene - sesquiterpenes - accumulation
Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.
Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua
Bertea, C.M. ; Freije, J.R. ; Woude, H. van der; Verstappen, F.W.A. ; Perk, L. ; Marquez, V. ; Kraker, J.W. de; Posthumus, M.A. ; Jansen, B.J.M. ; Groot, Æ. de; Franssen, M.C.R. ; Bouwmeester, H.J. - \ 2005
Planta Medica 71 (2005)1. - ISSN 0032-0943 - p. 40 - 47.
dihydroartemisinic acid - amorpha-4,11-diene synthase - chicory - roots
An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the - so far unknown - amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid