The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

Abstract

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the ⌬11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde ⌬11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small ␣,␤-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.Since its discovery in the 1970s (1), the sesquiterpene lactone artemisinin (Fig. 1) from Artemisia annua has become a key factor in the drive to control malaria (2-4). Semi-synthetic derivatives of plant-derived artemisinin form the basis for artemisinin-based combination therapies, which are the treatment of choice for most forms of the disease. Given the pharmaceutical importance of artemisinin and its singular plant source, there is considerable interest in maintaining a reliable and low cost supply (5). A more complete knowledge of artemisinin biosynthesis and the genes involved is likely to provide ways of increasing production and lowering cost through crop improvement or microbial engineering (6, 7).As in other members of the Asteraceae, A. annua has 10-celled biseriate glandular trichomes that appear on the surfaces of aerial parts of the plant (8 -10). Along with other isoprenoids, the sesquiterpene lactone artemisinin accumulates to levels of 0.01-2% dry weight (11). Although progress is being made in understanding the biosynthesis of artemisinin, considerable gaps in our knowledge remain (6, 12). The formation of amorpha-4,11-diene by amorpha-4,11-diene synthase is the first committed step in the pathway. This is followed by oxidation at C-12 of amorpha-4,11-diene by the cytochrome P-450, Cyp71av1 to give artemisinic alcohol. These steps are well supported from biochemical studies and by the molecular cloning of genes encoding the relevant enzymes (12-14). The pathway beyond artemisinic alcohol is somewhat less well established (6, 7, 15). However, there is biochemical evidence supporting a route to dihydroartemsinic acid via artemisinic aldehyde and dihydroartemsinic aldehyde (12). This route includes the proposed reduction of the ⌬11(13) double bond of artemisinic aldeh...

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

“…Although the enzymatic steps involved in production of the nonphytotoxic precursors amorpha-4,11-diene (A-4,11-D) and dihydroartemisinic acid (DHAA) have been elucidated (12)(13)(14)(15) and the associated genes have been shown to be highly expressed in both the apical and subapical cells of the glandular secretory trichomes (3,16), the final steps in the conversion of DHAA to artemisinin are considered to be nonenzymatic and may be extracellular (17,18). Therefore, microbial-based "complete" synthetic biology routes to artemisinin may never be achievable.…”

A glandular trichome-specific monoterpene alcohol dehydrogenase from Artemisia annua

“…The expression pattern matches the presence of ESTs corresponding to ADH2 from flower bud (AAFB), glandular trichome (AAGST), and "trichome-minus-bud" (GSTSUB) libraries (see 2.1). This expression pattern strongly suggests a role in terpenoid biosynthesis for ADH2 and has been seen for other enzymes proposed to have functions in trichome-specific terpenoid biosynthesis in A. annua (Teoh et al, 2009, Zhang et al, 2008.…”

“…The major components of A. annua essential oil are monoand sesquiterpenes (Ma et al, 2007), and they are thought to be biosynthesized within glandular trichomes (Duke et al, 1993, Olsson et al, 2009, Tellez et al, 1999. The sesquiterpenes in A. annua, in particular, the anti-malarial compound artemisinin and related compounds, have been studied extensively (Bertea et al, 2005, Covello et al, 2007, Ro et al, 2006, Teoh et al, 2006, Zhang et al, 2008. The proportion of the major essential oil components varies widely in different lines (or ecotypes) of A. annua.…”

“…annua (Covello et al, 2007, Covello, 2008, Teoh et al, 2009, Zhang et al, 2008. Indeed some of the largest contigs in the trichome-derived EST collection, i.e., the ones representing high expression, correspond to genes involved in isoprenoid biosynthesis (see Table 1).…”

Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae)

“…According to the reasonable extent of the artemisinin biosynthetic pathway that is known, more and more results point to dihydroartemisinic acid as the precursor of artemisinin. Therefore, artemisinic aldehyde reductase (DBR2) (Zhang et al, 2008), which catalyzes the conversion of artemisinic aldehyde to dihydroartemisinic aldehyde, and aldehyde dehydrogenase homologue (ALDH1) (Teoh et al, 2009), which converts dihydroartemisinic aldehyde to dihydryoartemisinic acid, seem more important in artemisinin biosynthesis. To increase artemisinin in A. annua, we believe that in co-overexpressing the combination of artemisinin biosynthesis genes, fps+dbr2, ads+dbr2, fps+aldh1, and ads+aldh1, for example, should be good candidates.…”