Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annuaThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Abstract

Limitations in the supply of the antimalarial compound artemisinin from Artemisia annua L. have led to an interest in understanding its biosynthesis and enhancing its production. Recent biochemical and molecular genetic data have implicated dihydroartemisinic aldehyde as a precursor to the corresponding acid, which is then converted to artemisinin. Thus, it is important to understand the enzyme or enzymes involved in dihydroartemisinic aldehyde oxidation. Given its activity on artemisinic aldehyde, the cytochrome P450 CYP71AV1 was investigated for its ability to oxidize dihydroartemisinic aldehyde. However, no net activity was detected. In a search for alternative enzymes that could catalyze the oxidation, an expressed sequence tag (EST) collection from A. annua was investigated for relevant cDNAs. This led to the isolation of a full-length cDNA encoding an aldehyde dehydrogenase homologue, named Aldh1, which is highly expressed in trichomes. Expression of the cDNA in E. coli and characterization of the purified recombinant enzyme revealed that the gene product catalyses the NAD(P)-dependent oxidation of the putative artemisinin precursors, artemisinic and dihydroartemsinic aldehydes, and a limited range of other aldehydes. The observed enzyme activity of Aldh1 and the expression pattern of the corresponding gene suggest a role in artemisinin biosynthesis in the glandular secretory trichomes of A. annua.

Relative expression of genes of terpene metabolism in different tissues of Artemisia annuaL

“…The next enzyme, CYP71AV1, was cloned by two groups independently [12,38]. The following two enzymes, DBR2 and ALDH1, were cloned and characterized by Zhang et al [15] and Teoh et al [16], respectively. Finally, RED1 was cloned and characterized by Rydén et al [18].…”

“…Dihydroartemisinic acid is formed from artemisinic aldehyde in two steps via dihydroartemisinic aldehyde. The reduction is catalyzed by artemisinic aldehyde Δ11(13) reductase (DBR2) [15] and the oxidation to the acid by aldehyde dehydrogenase 1 (ALDH1) [16]. It has not been fully evaluated if the CYP71AV1 enzyme can catalyze the oxidation of dihydroartemisinic aldehyde to the corresponding acid [16,17].…”

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination <i>Artemisia annua</i> Progeny and Artemisinin Formation in Regenerated Plants

“…For example, genetic evidence from Arabidopsis thaliana and other model plants has helped the intensive understanding of anthocyanin and proanthocyanidin pathways in the plant kingdom [50][51][52][53][54]. To date, biochemical, molecular and synthetic evidence has demonstrated the enzymatic steps from amorphor-4, 11-diene to artemisinic acid and dihydroartemisinic acid [22][23][24][25][26]55] and mapping of F1 hybrid of A. annua has helped identify loci associated with artemisinin formation [5], however, genetic evidence, such as knockout of genes and their impact on artemisinin productions, remains largely lacking. One of crucial reasons has been the challenge of the heterogeneous progeny resulting from the cross-hybridization preference of A. annua [2,9,27,28].…”

Transcriptome-Guided Mining of Genes Involved in Crocin Biosynthesis

“…A neighbor-joining (NJ) tree was constructed using a Poisson model with 1,000 bootstrap replicates. The protein sequences included CCD1, CCD4a, NCED1, NCED2, ALDH12, ALDH14, UGT60, UGT67, UGT86, and UGT89 from this study; AtCCD1, AtCCD4, AtNCED3, AtNCED5, AtNCED6, AtCCD7, AtCCD8, AtALDH2C4, AtALDH2B7 UGT73C6, and UGT73B2 from A. thaliana (Skibbe et al, 2002; Jones et al, 2003; Nair et al, 2004; Auldridge et al, 2006; Kim et al, 2006; Alder et al, 2012; Frey et al, 2012; Gonzalez-Jorge et al, 2013); CsCCD2, CsGT45, and UGTCs2 from C. sativus (Moraga et al, 2004; Frusciante et al, 2014); MtCCD1 and UGT73K1 from Medicago truncatula (Achnine et al, 2005; Floss et al, 2008; Moraga et al, 2009); CmCCD1 from Cucumis melo (Ibdah et al, 2006); CmCCD4a from C. morifolium (Ohmiya et al, 2006); CitCCD4 from C. unshiu (Ma et al, 2013; Rodrigo et al, 2013); CaALDH1 from C. annuum (Kim and Hwang, 2015); AaALDH1 from A. annua (Teoh et al, 2009); BoBADH from B. orellana (Bouvier et al, 2003); REF1 from B. napus (Mittasch et al, 2013); Zmrf2 (ALDH2B2) from Z. mays (Cui et al, 1996); BALDH from A. majus (Long et al, 2009); UGT75L6 and UGT94E5 from G. jasminoides (Nagatoshi et al, 2012); VLOGT1, VLOGT2, and VLOGT3 from Vitis labrusca (Hall et al, 2011); Gt5GT7 from Gentiana triflora (Nakatsuka et al, 2008); UGT1, UGTPg29, and UGTPg45 from P. ginseng (Yan et al, 2014; Wang et al, 2015); CrUGT8 from Catharanthus roseus (Asada et al, 2013); AdGT4 from Actinidia deliciosa (Yauk et al, 2014); GAME2 from Solanum lycopersicum (Itkin et al, 2013); ZOG1 from Phaseolus lunatus (Martin et al, 1999b); and ZOX1 from Phaseolus vulgaris (Martin et al, 1999a). Conserved motifs in CCDs, ALDHs and UGTs were detected using motif based sequence analysis tool MEME (Suite version 4.11.2).…”