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

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Δ11(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 Δ11(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 α,β-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. At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Δ11(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 Δ11(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 α,β-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 (1Liu J.-M. Ni M.-Y. Fan J. -F. Tu Y. -Y. Wu Z. -H. Wu Y. -L. Chou W.-S. Acta Chim. Sin. 1979; 37: 129-143Google Scholar), the sesquiterpene lactone artemisinin (Fig. 1) from Artemisia annua has become a key factor in the drive to control malaria (2Mutabingwa T.K. Acta Trop. 2005; 95: 305-315Crossref PubMed Scopus (236) Google Scholar, 3Rathore D. McCutchan T.F. Sullivan M. Kumar S. Expert Opin. Investig. Drugs. 2005; 14: 871-883Crossref PubMed Scopus (52) Google Scholar, 4Haynes R.K. Curr. Top. Med. Chem. 2006; 6: 509-537Crossref PubMed Scopus (206) Google Scholar). 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 (5Kindermans J.M. Pilloy J. Olliaro P. Gomes M. Malar. J. 2007; 6: 125Crossref PubMed Scopus (66) Google Scholar). 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 (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 7Zeng Q. Qiu F. Yuan L. Biotechnol. Lett. 2008; 30: 581-592Crossref PubMed Scopus (69) Google Scholar). 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 (8Wagner G.J. Wang E. Shepherd R.W. Ann. Bot. (Lond.). 2004; 93: 3-11Crossref PubMed Scopus (421) Google Scholar, 9Ferreira J.F.S. Simon J.E. Janick J. Hort. Rev. 1997; 19: 319-371Google Scholar, 10Duke S.O. Paul R.N. Int. J. Plant Sci. 1993; 154: 107-118Crossref Google Scholar). Along with other isoprenoids, the sesquiterpene lactone artemisinin accumulates to levels of 0.01–2% dry weight (11Duke M.V. Paul R.N. Elsohly H.N. Sturtz G. Duke S.O. Int. J. Plant Sci. 1994; 155: 365-372Crossref Scopus (224) Google Scholar). Although progress is being made in understanding the biosynthesis of artemisinin, considerable gaps in our knowledge remain (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 12Bertea C.M. Freije J.R. Van der Woude H. Verstappen F.W. Perk L. Marquez V. de Kraker J.W. Posthumus M.A. Jansen B.J. de Groot A. Franssen M.C. Bouwmeester H.J. Planta Med. 2005; 71: 40-47Crossref PubMed Scopus (188) Google Scholar). 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 (12Bertea C.M. Freije J.R. Van der Woude H. Verstappen F.W. Perk L. Marquez V. de Kraker J.W. Posthumus M.A. Jansen B.J. de Groot A. Franssen M.C. Bouwmeester H.J. Planta Med. 2005; 71: 40-47Crossref PubMed Scopus (188) Google Scholar, 13Chang Y.J. Song S.H. Park S.H. Kim S.U. Arch. Biochem. Biophys. 2000; 383: 178-184Crossref PubMed Scopus (123) Google Scholar, 14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar). The pathway beyond artemisinic alcohol is somewhat less well established (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 7Zeng Q. Qiu F. Yuan L. Biotechnol. Lett. 2008; 30: 581-592Crossref PubMed Scopus (69) Google Scholar, 15Li Y. Huang H. Wu Y.-L Liang X-T. Fang W.-S. Medicinal Chemistry of Bioactive Natural Products. John Wiley & Sons, Inc., New York2006: 183-256Google Scholar). However, there is biochemical evidence supporting a route to dihydroartemsinic acid via artemisinic aldehyde and dihydroartemsinic aldehyde (12Bertea C.M. Freije J.R. Van der Woude H. Verstappen F.W. Perk L. Marquez V. de Kraker J.W. Posthumus M.A. Jansen B.J. de Groot A. Franssen M.C. Bouwmeester H.J. Planta Med. 2005; 71: 40-47Crossref PubMed Scopus (188) Google Scholar). This route includes the proposed reduction of the Δ11(13) double bond of artemisinic aldehyde (Fig. 1). Artemisinin per se appears to be derived from dihydroartemisinic acid in a series of reactions that may not be enzyme-dependent (16Brown G.D. Sy L.-K Tetrahedron. 2004; 60: 1139-1159Crossref Scopus (134) Google Scholar, 17Sy L.K. Brown G.D. Tetrahedron. 2002; 58: 897-908Crossref Scopus (114) Google Scholar). Alternatively, based on labeling studies, artemisinic acid and derivatives, such as arteannuin B and artemisitene, have been suggested as precursors to artemisinin (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 7Zeng Q. Qiu F. Yuan L. Biotechnol. Lett. 2008; 30: 581-592Crossref PubMed Scopus (69) Google Scholar, 15Li Y. Huang H. Wu Y.-L Liang X-T. Fang W.-S. Medicinal Chemistry of Bioactive Natural Products. John Wiley & Sons, Inc., New York2006: 183-256Google Scholar). Clearly, a more detailed knowledge of the biochemistry of Δ11(13) double bond reduction during artemisinin formation is important in differentiating the proposed routes, which can be considered “early” and “late” reduction pathways. As part of an effort to understand the isoprenoid metabolism in glandular trichomes of the A. annua L., we have undertaken an expressed sequence tag (EST)-based 4The abbreviations used are: EST, expressed sequence tag; AA, artemisinic acid; Ads, amorpha-4,11-diene synthase; AtOPR, A. thaliana 12-oxophytodienoate reductase; Cpr, A. annua cytochrome P-450 reductase; Cyp71av1, amorpha-4;11-diene monooxygenase; Dbr2, artemisinic aldehyde Δ11(13) reductase; Fps2, A. annua farnesyl diphosphate synthase; GC/MS, gas chromatography/mass spectrometry; MES, 4-morpholineethanesulfonic acid; RT, reverse transcription; GFP, green fluorescent protein; OPR, 12-oxophytodienoate reductase; OYE, old yellow enzyme. approach to identify relevant genes (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar). This led to the identification of Cyp71av1, a multifunctional cytochrome P-450 capable of multiple oxidations of amorpha-4,11-diene. In a continuation of our investigation of the enzymes involved in artemisinin biosynthesis, we report here on the combined use of enzyme purification, mass spectrometry, and EST analysis leading to the molecular cloning and characterization of a sesquiterpenoid double bond reductase from A. annua. The characterization of this enzyme provides important support for the early Δ11(13) reduction in artemisinin biosynthesis. Plant Materials—A. annua seeds were obtained from Pedro Melillo de Magalhães (State University of Campinas, Brazil; line 2/39). The A. annua 2/39 line is characterized as a high artemisinin chemotype with a characteristically high dihydroartemisinic acid to artemisinic acid ratio (∼20:1 in flower buds; data not shown) (18Wallaart T.E. Pras N. Beekman A.C. Quax W.J. Planta Med. 2000; 66: 57-62Crossref PubMed Scopus (215) Google Scholar). The seeds were germinated and grown in soil in a controlled environment chamber with 16-h 24 °C days and 8-h 21 °C nights. Plants that had reached the height of ∼1.2 m (after about 3 months) were transferred to a flowering chamber with 8-h 24 °C days and 16-h 21 °C nights. Roots, leaves (mixtures of upper, middle, and lower leaves), and flower buds (27–29 days after flower induction under short days) were harvested and stored at -80 °C for subsequent RNA isolations and enzyme assays. Glandular trichomes were prepared from flower buds as described previously (14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar). Chemicals—Artemisinin, pyridinium chlorochromate, coniferyl aldehyde, 2-cyclohexen-1-one, 2E-hexenal, hexanal, 2E-nonenal, nonanal, (+)-α-pinene, and (+)-pulegone were obtained from Aldrich, and (+)-carvone, cyclohexanone, dihydrocarvone were obtained from Sigma. 12-Oxophytodienoic acid was obtained from Cedarlane Laboratories (Burlington, Canada). Arteannuin B and artemisitene were kindly provided by Dieter Deforce (University of Ghent). Artemisinic alcohol preparation was described previously (14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar). Sabinone was synthesized from sabinyl acetate obtained from the Plant Biotechnology Institute terpene collection (19von Rudloff E. Can J. Chem. 1963; 41: 2876-2881Crossref Google Scholar) by saponification of the sabinyl acetate to d-sabinol followed by oxidation of the alcohol using pyridinium chlorochromate (20Corey E.J. Suggs J.W. Tetrahedron Lett. 1975; 16: 2647-2650Crossref Scopus (2701) Google Scholar). The product was verified by mass spectral analysis. Isolation and/or semi-synthesis of artemisinic acid and artemisinic aldehyde, dihydroartemisinic acid, and three preparations containing dihydroartemisinic aldehyde epimers were prepared as described in the supplemental data. GC/MS Analysis—GC/MS analyses were performed on an Agilent 6890N gas chromatograph coupled to an Agilent 5973N mass selective in with an for sesquiterpenoid analysis or a & for other The was from to °C at °C the was at an of °C for and to °C at °C GC/MS analysis of reductase using arteannuin B and artemisitene was using a short with to °C to of and an of °C at °C of a of artemisinin was double bond reductase were as The reactions were by to a containing and of enzyme in enzyme preparations was by protein were with and The reactions were to for at °C with by of acid, and with The acetate extracts were used for GC/MS analysis. The were by and data with of 3 of was used as an of the reductase in plant the partial of the and protein the supplemental data. analysis of acid similarity to Dbr2 was performed as described previously D. Reed D.W. Covello P.S. Qiu J. Chem. 2007; PubMed Scopus Google Scholar). of Recombinant Artemisinic Δ11(13) Dbr2 was prepared as described in the supplemental data. characterization of the the were to and the reactions were by of The of the Dbr2 was to be in that of three and to and in and artemisinic aldehyde and of were under that to less as of artemisinic aldehyde of Dbr2, of and were in the of of of Dbr2, and of were in the of artemisinic The acetate extracts from were by was used as an to the from the reactions using using for enzyme The and were by analysis using Inc., RNA Isolation and Expression RNA was from plant using a Plant RNA analysis of Dbr2 first cDNA was prepared using reverse using of RNA as the was performed using a with a The were as °C for °C for followed by of °C for °C for and °C for and of °C for °C for and °C for The and were used to a of the Dbr2 The and were used to a of a gene A. annua by the These were based on the sequence of a cDNA corresponding to A. annua from an product using the and and A. annua of the of artemisinic aldehyde Δ11(13) Dbr2 and thaliana were the The obtained from Biotechnology was derived from M. D. A. Plant Sci. 2002; PubMed Scopus Google Scholar) by the of a sequence encoding of the and a sequence encoding an of the The of A. thaliana was first obtained by using the and The product was using to give The of Dbr2 and were using reactions with and to give the and were transferred to the for of A. thaliana using a by and was by after which were for days in and to of the was with a with with a and was in a with and for and was in the via a and was via a preparation of the and and use in yeast are described in the supplemental data. Artemisinic Δ11(13) in A. annua of the enzyme involved in the sesquiterpenoid Δ11(13) double bond reduction was investigated in extracts of A. annua 2/39 with flower buds reductase activity in the of artemisinic aldehyde and (Fig. and This resulted in the of a product by GC/MS that was as aldehyde (Fig. by with (Fig. and (Fig. B and preparations of dihydroartemisinic aldehyde This had a (Fig. and mass to aldehyde synthesized from dihydroartemisinic acid (Fig. and supplemental The was from the in the in the reduction of (Fig. the of artemisinin at the of the enzyme activity found is with a in artemisinin biosynthesis. for the reductase were and supplemental and extracts from flower and glandular trichomes were The for the four are shown in with reductase levels in extracts of flower buds and leaves were at In reductase activity was Given the of glandular trichomes on surfaces and at on the data from the plant extracts are with a enzyme. and of Artemisinic Δ11(13) from A. a complete characterization of the its from plant was buds were for in of the more glandular trichomes. Artemisinic aldehyde Δ11(13) reductase was from the flower buds of A. annua line 2/39 using and and supplemental data for This resulted in an on a protein basis to the The that the molecular weight of artemisinic aldehyde Δ11(13) reductase was not The enzyme was to followed by The most were from a with and by the data from a corresponding to was used to a data of A. annua the was to a cDNA clone derived from the A. annua trichome-specific cDNA (14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar). The mass spectral data to the four and The gene corresponding to this EST was of A. annua (14Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Covello P.S. FEBS Lett. 2006; 580: 1411-1416Crossref PubMed Scopus (334) Google Scholar) the of and corresponding to Dbr2 derived from flower glandular and This suggested a relatively high of Dbr2 in trichomes to flower Isolation of a Dbr2 cDNA from A. annua the characterization of the product of Dbr2, a cDNA clone was obtained by of cDNA The Dbr2 is and a acid protein with a molecular mass of A of with the acid protein sequence of Dbr2 sequence with plant 12-oxophytodienoate and sequence data relevant to this can be found in the under the A. thaliana and A. annua by amorpha-4,11-diene synthase A. annua cytochrome P-450 reductase Cyp71av1 Dbr2 by and H. S. S. and Z. and In Dbr2 high acid sequence to and of which have been shown to the 12-oxophytodienoate involved in biosynthesis J. F. N. P. A. Plant J. 2002; PubMed Scopus Google Scholar). the of analysis of Dbr2 and a of based on acid sequence Dbr2 a that includes the and as well as an from and the and on sequence there is support for of Dbr2 in the family of T.E. D.W. J. 2005; 58: PubMed Scopus Google Scholar), which old yellow enzymes J. J. J. 1994; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). This protein family is as the enoate analysis that Dbr2 and at and The are highly in and enzymes and are thought to be involved in in the of H. A. J. P. Sci. S. A. 2006; PubMed Scopus Google Scholar) were found in corresponding of Dbr2 and However, the that form a the of H. A. J. P. Sci. S. A. 2006; PubMed Scopus Google Scholar), are by in Dbr2 supplemental The in the to the are thought to be for the in of and H. A. J. P. Sci. S. A. 2006; PubMed Scopus Google Scholar). In the that most likely for the of and J. F. N. P. A. Plant J. 2002; PubMed Scopus Google Scholar, Plant PubMed Google Scholar) is with in the of This a of Dbr2 in A. annua Recombinant Dbr2 Artemisinic Δ11(13) characterization of Dbr2, the enzyme was from the The of Dbr2 from and that the enzyme as a The Dbr2 protein was with followed by GC/MS analysis. to plant Dbr2 formation of aldehyde as the product using artemisinic aldehyde as a with enzyme preparation shown) and in the of not support the production of dihydroartemisinic Given the similarity of Dbr2 to and 12-oxophytodienoate reductase Dbr2 was for activity with acid. were found in this not Dbr2 was with other arteannuin artemisinic acid, artemisinic artemisitene, (+)-carvone, coniferyl aldehyde, 2-cyclohexen-1-one, 2E-hexenal, 2E-nonenal, (+)-α-pinene, and The in to artemisinic aldehyde, Dbr2 has activity on 2-cyclohexen-1-one, (+)-carvone, and low activity on of the for artemisinic supplemental The and mass of the of artemisinic aldehyde, (+)-carvone, 2-cyclohexen-1-one, and aldehyde, cyclohexanone, and and on with a of and that the at was the and of were as and which were in a activity was with arteannuin artemisinic acid, artemisinic artemisitene, coniferyl aldehyde, 2E-nonenal, (+)-α-pinene, and not The of Dbr2 was to be At the enzyme activity was at of its activity at that the enzyme was in the of were for artemisinic aldehyde, 2-cyclohexen-1-one, (+)-carvone, and to and (+)-carvone, the reductase was specific for artemisinic aldehyde, for which the was more lower 1). The enzyme is highly specific for with a of as with more for for in a Dbr2 Expression and of Dbr2 in A. annua was using As in Dbr2 is relatively high in glandular less in leaves and flower and in This is to the reductase in (Fig. and the in of Dbr2 in artemisinin biosynthesis in glandular trichomes. The of Dbr2 was investigated by of in A. thaliana The Dbr2 protein to the (Fig. the protein to the the importance of the sequence as a T.E. D.W. J. 2005; 58: PubMed Scopus Google Scholar). of in of Dbr2 the engineering of for the production of This was by of multiple genes in In dihydroartemisinic aldehyde to yeast was to dihydroartemisinic acid, as a of aldehyde activity not on was that the de production of dihydroartemisinic acid be by of farnesyl diphosphate synthase amorpha-4,11-diene amorpha-4,11-diene cytochrome P-450 and artemisinic aldehyde Δ11(13) reductase in three yeast were a control containing three a that the artemisinin pathway to artemisinic acid by Fps2, amorpha-4,11-diene Cyp71av1, and A. annua cytochrome P-450 reductase; and a that As in the control the through Cyp71av1 not artemisinic acid to a of This is with levels found in previously M. J.M. J. M.C. Y. 2006; PubMed Scopus Google Scholar). In the Dbr2, artemisinic acid to and in dihydroartemisinic acid was found at a of The molecular cloning of Dbr2 is an of the of the combined use of protein purification, mass spectrometry, and expressed sequence tag analysis. In the of and biochemical knowledge the identification of an enzyme using relatively protein mass the to be made of a protein and corresponding cDNA in this The understanding of the biosynthesis of artemisinin is somewhat (6Covello P.S. Teoh K.H. Polichuk D.R. Reed D.W. Nowak G. Phytochemistry. 2007; 68: 1864-1871Crossref PubMed Scopus (141) Google Scholar, 7Zeng Q. Qiu F. Yuan L. Biotechnol. Lett. 2008; 30: 581-592Crossref PubMed Scopus (69) Google Scholar, 15Li Y. Huang H. Wu Y.-L Liang X-T. Fang W.-S. Medicinal Chemistry of Bioactive Natural Products. John Wiley & Sons, Inc., New York2006: 183-256Google Scholar). data remain in the of the of has suggested that such as artemisinic acid, arteannuin artemisitene, and are However, other evidence the that artemisinic acid and dihydroartemisinic acid in the with the to This is supported by in (12Bertea C.M. Freije J.R. Van der Woude H. Verstappen F.W. Perk L. Marquez V. de Kraker J.W. Posthumus M.A. Jansen B.J. de Groot A. Franssen M.C. Bouwmeester H.J. Planta Med. 2005; 71: 40-47Crossref PubMed Scopus (188) Google Scholar) and in biochemical studies (16Brown G.D. Sy L.-K Tetrahedron. 2004; 60: 1139-1159Crossref Scopus (134) Google Scholar). A key in the proposed to artemisinin is the point at which the Δ11(13) double bond is reduced. In the early reduction has been suggested to occur in the of artemisinic aldehyde to dihydroartemisinic the other reduction Δ11(13) reduction at artemisinic acid, arteannuin or artemisitene or derivatives Q. Qiu F. Yuan L. Biotechnol. Lett. 2008; 30: 581-592Crossref PubMed Scopus (69) Google Scholar). The characterization of Dbr2 has on the of the pathway to In that artemisinic alcohol is a to artemisinin, the of Dbr2 for artemisinic aldehyde artemisinic alcohol and acid, as well as arteannuin B and artemisitene, is support for the early reduction pathway. have such an enzyme highly expressed in trichomes that at some artemisinin is made via its dihydroartemisinic aldehyde, and subsequently dihydroartemsinic acid. that a reduction pathway is in at appears that such a route may be is that have been A. annua that in artemisinin and the (18Wallaart T.E. Pras N. Beekman A.C. Quax W.J. Planta Med. 2000; 66: 57-62Crossref PubMed Scopus (215) Google Scholar). artemisinin to have high levels of artemisinic acid; high artemisinin to have high dihydroartemsinic acid. is of to that in Dbr2 is for this and is in progress to In that Dbr2 activity may be a factor in artemisinin biosynthesis, an important for engineering artemisinin this in can that Dbr2 be for of high of Dbr2 and for the of for high Dbr2 be to in the of artemisinin The biochemistry of double bond reduction includes enzymes that on α,β-unsaturated has been investigated the carbonyl is thought to be involved in the This is of the family to which Dbr2 which includes the old yellow enzyme 2002; PubMed Scopus Google Scholar). by and be to bond to the carbonyl of artemisinic aldehyde H. A. J. P. Sci. S. A. 2006; PubMed Scopus Google Scholar). and to the and are likely to and of the first terpene double bond characterized were the glandular trichome-specific and (+)-pulegone from Arch. Biochem. Biophys. PubMed Scopus Google Scholar). These enzymes are members of the short and Dbr2 a with terpene double bond the this the enzymes most to Dbr2 are the plant of and on the α,β-unsaturated acid as part of the pathway to acid. However, the in of most other plant genes is In the sesquiterpenoid reductase activity of Dbr2 appears to be of the first of an activity other acid reduction that can be to an in pathway. This the that have been during for in about the of other in the The cloning and characterization of Dbr2 some In this we have a for the microbial production of dihydroartemisinic acid. the of A. annua genes in we were to dihydroartemisinic acid production at levels with previously for artemisinic acid M. J.M. J. M.C. Y. 2006; PubMed Scopus Google Scholar). In the sesquiterpene acid can be to However, the of dihydroartemisinic acid to artemisinin is under the are to Polichuk for and for Dieter and for and and for the with

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