artemisinic acid was isolated from dichloromethane extracts of a . annua flower buds and leaves and was used to synthesize artemisinic aldehyde according to the method described by chang et al . 2000 , the disclosure of which is incorporated herein by reference . dihydroartemisinic acid was isolated and purified from a . annua leaf material obtained from a “ line 2 / 39 ” containing relatively high levels of the dihydroartemisinic acid using the method described for artemisinic acid in teoh et al . 2006 , the disclosure of which is incorporated herein by reference . dihydroartemisinic aldehyde was synthesized from the isolated dihydroartemisinic acid . the acid was converted to methyl dihydroartemisinate with excess diazomethane in diethyl ether at 0 ° c . for 5 minutes . the ether and diazomethane were removed under a stream of nitrogen and the methyl ester was reduced to ( 11r )- dihydroartemisinic alcohol with excess 1 . 5 m diisobutyl aluminum hydride in toluene at room temperature for 10 min under nitrogen . with subsequent extraction , oxidation to the aldehyde with pyridinium chlorochromate ( corey & amp ; suggs 1975 ) and purification by hplc the ( 11r )- dihydroartemisinic aldehyde was produced at an overall yield of 48 % with & gt ; 99 % purity according to gc analysis . artemisia annua l . seeds were obtained from elixir farm botanicals , brixey , mo ., usa and from pedro melillo de magalhães , state university of campinas , brazil ( line 2 / 39 ). seeds were germinated and grown in soil in a controlled environment chamber with 16 hour / 25 ° c . days and 8 hour / 20 ° c . nights . plants that had reached the height of approximately 1 . 2 m ( about 3 months ) were transferred to flowering chamber with 12 hour / 25 ° c . days and 12 hour / 20 ° c . nights . flower buds that developed after 19 - 21 days in the flowering chamber were harvested for total rna isolation . total rna was extracted and isolated from glandular trichomes and flower buds using a modified method described by logeman , et al . 1987 . cdna synthesis from 1 . 5 micrograms of total rna and construction of the trichome and flower bud cdna library were carried out with creator ™ smart ™ cdna library construction kit ( clontech ). a total of 6 , 239 clones and 2 , 208 clones for trichome and flower bud libraries , respectively were randomly picked and their dna sequences determined . sequencing was performed on an ab13700 dna sequencer using bigdye terminator cycle sequencing kit ( applied biosystems ) and the m13 reverse primer . dna sequence traces were interpreted and vector and low quality sequences were eliminated using phred ( ewing et al . 1998 ) and lucy ( chou & amp ; holmes 2001 ). clustering of the resulting est dataset was done using stackpack ( miller et al . 1999 ) and sequence similarity was identified by blast ( altschul et al . 1990 ). the open reading frame ( orf ) of a double bond reductase designated aadbr1 , encoded by the est clone pkt104 , was obtained through pcr using gene - specific primers 5 ′- caccatggaacagcaacmgaag - 3 ′ ( seq id no . : 9 ) and 5 ′- tcattcatgcgcaaccaccacca - 3 ′ ( seq id no . : 10 ) and vent polymerase ( new england biolabs , cambridge , mass ., usa ). the resulting pcr product was cloned via the gateway entry vector pentr / d / topo ( invitrogen ) into a gateway destination vector , pdest17 ( invitrogen ) to generate a bacteria expression clone pkt032 . the orf of aadbr1 was cloned in frame with the 6 × his - tag ( seq id no . : 13 ) at the n - terminal of aadbr1 . the open reading frame ( orf ) of an aldehyde dehydrogenase designated aaaldh1 , encoded by the est clone pkt150 , was obtained through pcr using gene - specific primers 5 ′- caccatgagctcaggagctaat - 3 ′ ( seq id no . : 11 ) and 5 ′- ttaaagccacggggaatcatat - 3 ′ ( seq id no . 12 ) and vent polymerase ( new england biolabs , cambridge , mass ., usa ). the resulting pcr product was cloned via the gateway entry vector pentr / d / topo ( invitrogen ) into a gateway destination vector , pdest17 ( invitrogen ) to generate a bacterial expression clone pkt041 . the orf of aaaldh1 was cloned in frame with the 6 × his - tag ( seq id no . : 13 ) at the n - terminal of aaaldh1 . the plasmid pkt032 or pkt041 was introduced into e . coli strain bl21 ( de3 ) ( novagen ) using heat shock at 42 ° c . the gus gene ( invitrogen ) was cloned into pdest17 to replace the ccdb gene and the construct pdest - gus introduced into the e . coli strain bl21 ( de3 ) was used as a control . transformants were grown on luria broth ( lb ) and selected on ampicillin ( 100 μg / ml ) at 37 ° c . for 24 hours . a single colony containing pkt032 or pkt041 was used to inoculate 5 ml of lb liquid medium with ampicillin ( lba ) and grown at 37 ° c . overnight with shaking . the overnight culture was used to inoculate 250 ml of lba liquid medium and grown at 37 ° c . with shaking to an od 600 of 0 . 6 per ml followed by induction with 1 mm iptg and grown at 30 ° c . overnight with shaking . cells were pelleted at 2 , 000 g at 4 ° c . for 10 minutes . the pelleted cells were resuspended in 6 ml of lysis buffer consisting of 50 mm sodium phosphate , ph 8 . 0 , 0 . 1 m nacl , 20 mm imidazole and 1 mm phenylmethylsufonyl fluoride ( pmsf ). cells were lysed with lysozyme ( 0 . 2 mg / ml of cells ) on ice for 30 minutes followed by sonication on ice with 30s pulse ( 5 ×). protein concentration was determined by bradford assay ( bio - rad ). the aadbr1 protein was detected by silver stain on sds gel and confirmed by western blot using anti - his antibody ( invitrogen ). the aaaldh1 protein was detected by rapid stain ( bioscience , st . louis , mo .) on sds gel . cell - free extract of recombinant aaaldh1 was prepared as described above . the cell - free extract was centrifuged at 20 , 000 g at 4 ° c . for 15 minutes to remove any remaining insoluble materials before loading onto a his - trap ff column ( amersham bioscience , n . j .) equilibrated with binding buffer ( 20 mm sodium phosphate buffer containing 500 mm nacl and 20 mm imidazole at ph 7 . 5 ). the column was washed with 5 column volume of binding buffer and the recombinant aaaldh1 eluted with elution buffer ( 20 mm sodium phosphate , 500 mm nacl , ph 7 . 5 ) containing increasing concentration of imidazole in a step - wise fashion . the eluted fractions were concentrated and desalted in centrifugal filter devices ( amicon ultra — 15 ) ( millipore , mass .) following manufacturer &# 39 ; s protocol . the purity of the recombinant aaaldh1 was checked with sds gel stained with rapid stain ( biosciences , st . louis , mo .). cell - free extracts of recombinant his - tagged aadbr1 protein were assayed with artemisinic aldehyde , followed by analysis by gas chromatography / mass spectrometry . enzyme reactions were initiated by adding the substrate ( 5 μg ) to 500 μl sodium phosphate buffer ( 50 mm , ph 7 . 5 ) containing 10 % sorbitol , 1 mm nadph , 2 mm dtt and 0 . 8 μg of enzyme . negative controls were carried out with boiled proteins , without nadph and with extracts from e . coli into which the construct pdest17 - gus had been introduced . reactions were allowed to proceed for 30 minutes at 30 ° c . with shaking and immediately stopped by extracting twice with 700 μl diethyl ether . the ether extracts were pooled , evaporated and taken up in 20 μl ethyl acetate ( sigma ) followed by gc - ms analysis . cell - free extract of recombinant his - tagged aaaldh1 protein were assayed with dihydroartemisinic aldehyde and other substrates , followed by analysis by gas chromatography / mass spectrometry . enzyme reactions were initiated by adding the substrate ( 5 μg ) to 500 μl tris - hcl buffer ( 50 mm , ph 8 . 5 ) containing , 1 mm nadp , and 1 . 0 μg of enzyme . negative controls were carried out with boiled proteins , without nadp and with extracts from e . coli into which the construct pdest17 - gus had been introduced . reactions were allowed to proceed for 30 minutes at 30 ° c . with shaking and immediately stopped by extracting twice with 700 μl diethyl ether . the ether extracts were pooled , derivatized with diazomethane , evaporated and taken up in 20 μl dichloromethane ( sigma ) followed by gc - ms analysis . the linearity of the assay with respect to time and protein concentration was first established and the operational saturation of substrate and cofactor determined . the ph optimum was determined by the standard assay in 50 mm buffer ( sodium phosphate , tris - hcl and ches ) from ph 6 . 0 to 10 . 0 at 0 . 5 unit interval containing 1 mm nadp and 1 . 5 micrograms of the purified recombinant aaaldh1 . kinetic parameters were determined in 50 mm tris - hcl buffer , ph 8 . 5 by varying the concentration of the substrates . kinetic constants were determined by non - linear regression analysis using graphpad software ( graphpad software inc . san diego , calif .) and the results presented are the means of three independent experiments . substrates specificity was determined at optimum reaction conditions with substrates concentration at 10 times the estimated km value . substrates tested include artemisinic aldehyde , ( 11r )- dihydroartemisinic aldehyde , artemisinic alcohol , dihydroartemisinic alcohol , octanal , nonanal , 2 - phenyl propionaldehyde , 3 - cyclohexyl propionaldehyde , 2 - hexen - 1 - al , syringaldehyde . expressed sequence tags ( sequences of randomly picked cdna clones ) were generated from developing trichomes and flower buds of artemisia annua and analyzed . cdna clones with sequences similar to monoterpene double - bond reductases were resequenced and these sequences were assembled . these were deemed to be derived from a single artemisia annua gene which was called aadbr1 . the consensus nucleotide sequence of the aadbr1 mrna is shown in fig2 ( seq id no . : 1 ). the corresponding amino acid sequence is shown in fig3 ( seq id no . : 2 ). cdna clones with sequences similar to aldehyde dehydrogenase were resequenced and these sequences were assembled . these were deemed to be derived from a single artemisia annua gene which was called aaaldh1 . the consensus nucleotide sequence of the aaaldh1 mrna is shown in fig6 ( seq id no . : 5 ). the corresponding amino acid sequence is shown in fig7 ( seq id no . : 6 ). for initial functional studies of aadbr1 , an rt - pcr product was prepared and cloned into e . coli expression vector pdest17 to give the clone pkt032 . the nucleotide sequence of the open reading frame of the dna insert of pkt032 is given in fig4 ( seq id no . : 3 ) and the corresponding protein product including the n - terminal his tag fusion is given in fig5 ( seq id no . : 4 ). the plasmid pkt032 was introduced into the e . coli ( de3 ) strain ( novagen ) and cell - free extracts was assayed with various isoprenoid substrates followed by analysis by gas chromatography / mass spectrometry . fig1 shows the results of this analysis indicating the nadph - dependent formation of ( 11s )- dihydroartemisinic aldehyde as the major product . in a separate experiment , extracts from e . coli into which pkt032 had not been introduced did not support the production of dihydroartemisinic aldehyde in the presence of nadph . it is predicted that the wild type product of aadbr1 will have similar artemisinic aldehyde double bond reductase activity as the his tag fusion protein product of pkt032 . for initial functional studies of aaaldh1 , a pcr product was prepared and cloned into e . coli expression vector pdest17 to give the clone pkt041 . the nucleotide sequence of the open reading frame of the dna insert of pkt041 is given in fig8 ( seq id no . : 7 ) and the corresponding protein product including the n - terminal his tag fusion is given in fig9 ( seq id no . : 8 ). the plasmid pkt041 was introduced into the e . coli ( de3 ) strain ( novagen ) and cell - free extracts was assayed with ( 11r )- dihydroartemisinic aldehyde followed by analysis by gas chromatography / mass spectrometry . the recombinant aaaldh1 protein was purified from the cell - free extract and its kinetic parameters were determined . the purified recombinant aaaldh1 protein functions best at ph 8 . 5 . the recombinant protein was tested with different substrates ( see materials and methods ) at the optimum assay conditions . artemisinic aldehyde besides ( 11r )- dihydroartemisinic aldehyde was found to be a substrate for the recombinant aaaldh1 . the k m and v max values determined for dihydroartemisinic acid were 8 . 79 μm and 143 . 8 pkat / μg protein , respectively and for artemisinic aldehyde the km and v max were 2 . 62 μm and 28 . 6 pkat / μg protein , respectively . fig1 shows the results of the analysis for ( 11r )- dihydroartemisinic aldehyde indicating the nadp - dependent formation of dihydroartemisinic acid . in separate experiment , extracts from e . coli into which pkt041 had not been introduced did not support the production of dihydroartemisinic acid in the presence of nadp . it is predicted that the wild type product of aaaldh1 will have similar artemisinic / dihydroartemisinic aldehyde dehydrogenase activity as the his - tag fusion protein product of pkt041 . the disclosures of the following references are incorporated herein by reference in their entirety . altschul , s . f ., gish , w ., miller , w ., myers , e ., & amp ; lipman , d . j . 1990 , “ basic local alignment search tool ”, journal of molecular biology , vol . 215 , pp . 403 - 410 . bagge , m ., xia , x ., & amp ; lubberstedt , t . 2007 , “ functional markers in wheat ”, curr . opin . plant biol ., vol . 10 , no . 2 , pp . 211 - 216 . bertea , c . m ., freije , j . r ., van der , w . h ., verstappen , f . w ., perk , l ., marquez , v ., de kraker , j . w ., posthumus , m . a ., jansen , b . j ., de groot , a ., franssen , m . c ., & amp ; bouwmeester , h . j . 2005 , “ identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in artemisia annua ”, planta med ., vol . 71 , no . 1 , pp . 40 - 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