The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage

Abstract

SummaryArtemisia annua, which produces the anti-malaria compound artemisinin, occurs as highartemisinin 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.

Natural products – modifying metabolite pathways in plants

“…In addition, the tissue-specific expression profiles thus obtained may be further correlated with the expected product accumulation profiles of the tissue in question. In this manner, we have recently identified and characterized the genes of the costunolide [17] and artemisinin biosynthetic pathways [18,19].…”

“…The pathway genes were also stably transformed into tobacco, resulting in trace artemisinin detection [34]. Furthermore, transient expression of all known artemisinin biosynthetic genes in N. benthamiana showed that changes in the relative activity of the individual steps can improve the flux to the dihydro artemisinic acid (DHAA) segment of the pathway, ultimately leading to improved artemisinin production [18]. As the conversion mechanism of dihydroartemisinic acid to artemisinin in planta remains elusive, the full potential for production of the valuable antimalarial in heterologous plant systems has not yet been satisfactorily explored.…”

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

“…CYP71AV1 in the low artemisinin-chemotype has extra amino acid residues in its N-terminal, whereas CYP71AV1 in the high-artemisinin chemotype has not such extension. Upon transient expression in N. benthamiana, high artemisinin-chemotype CYP71AV1 exhibits higher enzyme activity than low artemisinin-chemotype CYP71AV1 (Ting et al 2013).…”

“…Nevertheless, the multiple enzymatic routes to artemisinic acid and dihydroartemisinic acid have been validated, as illustrated in Figure 1 (Ting et al 2013). Overall, intermediates were identified and assigned sequentially, enzymes were purified and structurally/functionally characterized, and encoding genes were cloned and homologously/ heterologously expressed.…”

“…ADS, amorphadiene synthase; ALDH1, aldehyde dehydrogenase 1; CYP71AV1, amorphadiene oxidase; DBR2, artemisinic aldehyde double-bond reductase; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; RED1, dihydroartemisinic aldehyde reductase 1. Broken arrows indicate reactions more than one step (Ting et al 2013 with modifications).…”