Based on the content of artemisinin and its precursors, two chemotypes of Artemisia annua, can be distinguished: the low-artemisinin production (LAP) chemotype and a high-artemisinin production (HAP) chemotype (Wallaart et al., 2000). Both chemotypes contain artemisinin and arteannuin B, but the HAP chemotype has a relatively high content of artemisinin and its presumed precursor dihydroartemisinic acid (DHAA), while the LAP chemotype has a high content of arteannuin B and its presumed precursor artemisinic acid (AA).
The artemisinin biosynthesis pathway has been largely elucidated and the genes required for production of dihydroartemisinic acid, the most likely precursor of artemisinin (ADS, CYP71AV1, DBR2 and ALDH1) have all been described (Bouwmeester et al., 1999; Teoh et al., 2006, 2009; Zhang et al., 2008; Rydén et al., 2010). Artemisinin is a sesquiterpene lactone endoperoxide, which is synthesized in the cytosol from the general isoprenoid precursors IPP and DMAPP. These are converted to FPP and the first committed step in the artemisinin biosynthetic pathway is the cyclization of FPP to amorpha-4,11-diene (AD) by amorphadiene synthase (Fig. 1; Bouwmeester et al., 1999; Mercke et al., 2000). In the subsequent step, AD is oxidized by the cytochrome P450 enzyme, CYP71AV1/AMO, to artemisinic alcohol (AAOH), artemisinic aldehyde (AAA) and artemisinic acid (AA; Fig. 1; Ro et al., 2006; Teoh et al., 2006). However, the latter mainly occurs in the LAP chemotype. In the HAP chemotype only very little of the AAA is converted to AA, as most of the AAA is converted to dihydroartemisinic aldehyde (DHAAA) by DBR2, the enzyme that reduces the exocyclic double bond of AAA (Fig. 1; Bertea et al., 2005; Zhang et al., 2008). Supposedly, DHAAA is subsequently oxidized by alcohol dehydrogenase ALDH1 to the final intermediate dihydroartemisinic acid (DHAA; Bertea et al., 2005; Zhang et al., 2008). The conversion of DHAA to artemisinin, is believed to be a nonenzymatic and spontaneous photo-oxidation reaction (Wallaart et al., 1999; Sy & Brown, 2002). Similarly, in the LAP chemotype, AA is likely spontaneously converted to arteannuin B.
Figure 1. Artemisinin biosynthetic pathway in Artemisia annua. IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; AD, amorpha-4,11-diene; AAOH, artemisinic alcohol; AAA, artemisinic aldehyde; AA, artemisinic acid; AB, arteannuin B; DHAAOH, dihydroartemisinic alcohol; DHAAA, dihydroartemisinic aldehyde; DHAA, dihydroartemisinic acid; FPS, farnesyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; ADS, amorphadiene synthase; CYP71AV1, amorphadiene oxidase; DBR2, artemisinic aldehyde double-bond reductase; RED1, dihydroartemisinic aldehyde reductase 1; ALDH1, aldehyde dehydrogenase 1. Broken arrows indicate the involvement of more than one step (Nguyen et al., 2011).
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Recently we reported on the (transient) reconstruction of the artemisinin biosynthetic pathway in Nicotiana benthamiana leaves, resulting in up to 39.5 mg kg−1 FW of AA (van Herpen et al., 2010). In the present work we analyse the role of DBR2, ALDH1 and CYP71AV1 in determining the ‘chemotype’ (as defined by the AA and DHAA ratio) of N. benthamiana leaves agro-infiltrated with artemisinin biosynthesis genes. Results show that the chemotype is a function of the CYP71AV1 type and relative dosage of DBR2 and ALDH1.