Patent Application: US-73479108-A

Abstract:
an isolated nucleic acid molecule cloned from artemisia annua encodes an artemisinic aldehyde double bond reductase . artemisinic aldehyde double bond reductase enzymatically reduces artemisinic aldehyde to - dihydroartemisinic aldehyde . the nucleic acid molecule , and the enzyme encoded thereby , may be used in processes to produce dihydroartemsinic aldehyde and / or dihydroartemisinic acid in a host cell . dihydroartemisinic acid is a late precursor to the antimalarial compound artemisinin .

Description:
artemisinic acid was isolated from dichloromethane extracts of a . annua flower buds and leaves ( teoh , polichuk , reed , nowak , & amp ; covello 2006 ) 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 ( see above ). 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 ; 90 % purity according to gc analysis . artemisinin , pyridinium chlorochromate , coniferyl aldehyde , 2 - cyclohexen - 1 - one , 2e - hexenal , hexanal , 2e - nonenal , nonanal , (+)- α - pinene , and (+)- pulegone were obtained from aldrich . (+)- carvone , cyclohexanone , dihydrocarvone were obtained from sigma . arteannuin b and artemisitene were kindly provided by dieter deforce ( university of ghent ). artemisinic alcohol preparation was described previously ( teoh 2006 ). sabinone was synthesized from sabinyl acetate obtained from the plant biotechnology institute terpene collection ( von rudloff 1963 ) by saponification of the sabinyl acetate to d - sabinol followed by oxidation of the alcohol using pyridinium chlorochromate ( corey 1975 ). 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 for the elixir line and 8 hour / 25 ° c . days and 16 hour / 20 ° c . nights for line 2 / 39 . 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 ). preparation of the clarified extracts containing artemisinic aldehyde double bond reductase from artemisia annua all of the operations were carried out at 4 ° c . twenty four grams of flower buds from line 2 / 39 were ground in a waring blender with 100 ml of 0 . 1 m potassium phosphate ( ph 7 . 0 ) containing 5 mm dithiothreitol . the resulting slurry was filtered through three layers of cheesecloth . the filtrate was centrifuged at 10 , 000 × g for 15 min . the supernatant was precipitated in a step - wise fashion by the addition of ( nh 4 ) 2 so 4 . after removal of the precipitate at 30 % ( nh 4 ) 2 so 4 saturation , the precipitate of the supernatant at 80 % ( nh 4 ) 2 so 4 saturation was collected by centrifugation . the pellet was dissolved in 3 ml of 20 mm tris - hcl buffer ( ph 7 . 3 ) and centrifuged for 10 min at 10 , 000 × g . the clear supernatant was dialyzed against a tris - hcl buffer ( ph 7 . 3 ). at various stages of purification double bond reductase assays were performed on plant extracts with artemisinic aldehyde , followed by gas chromatography / mass spectrometry analysis . enzyme reactions were initiated by adding the 0 . 4 mm artemisinic aldehyde to 300 μl reaction mixture containing plant extract ( 10 - 300 μg protein ), 50 mm tris - hcl ( ph 8 . 0 ), 1 mm nadph and 2 mm dtt . negative controls were carried out with boiled proteins and without nadph . reactions were allowed to proceed for 30 minutes at 30 ° c . with shaking , stopped by the addition of 15 μl of acetic acid and extracted with 100 μl ethyl acetate . the ethyl acetate extracts were then subjected to gc / ms analysis . the reaction products were confirmed by comparing gc retention time and ms data with those of synthetic ( 11r )- dihydroartemisinic aldehyde . for quantification , octadecane was used as an internal standard . partial purification of the artemisinic aldehyde double bond reductase from artemisia annua the aforementioned dialyzed extract was applied to a mono - q hr strong anion ion exchange column ( 5 × 50 mm ; ge healthcare life sciences ) pre - equilibrated with 10 mm potassium phosphate buffer ( ph 7 . 8 ) containing 1 mm dtt and eluted with 30 ml of a linear kcl gradient ( 0 - 0 . 5 m ) in 10 mm potassium phosphate buffer ( ph 7 . 8 ) at a flow rate of 1 . 0 ml / min . one ml fractions were collected and tested for the artemisinic aldehyde double bond reductase activity as follows . each fraction was desalted by spin dialysis ( amicon ultra - 15 devices ; millipore , mass .) into assay buffer ( 50 mm potassium phosphate buffer , ph 7 . 5 ) containing 1 mm dtt . reactions were initiated with the addition of 1 mm nadph and 0 . 4 mm artemisinic aldehyde in a total volume of 100 μl , and allowed for proceed at 30 ° c . for 30 min prior to the addition of 50 μl ethyl acetate . thirty μl of ethyl acetate extracts from the reactions were used for gc - ms analysis . the reaction products were confirmed by comparing gc retention time and ms data those of synthetic ( 11r )- dihydroartemisinic aldehyde . for quantification octadecane was used as an internal standard . the active fractions were combined , desalted and concentrated by spin dialysis ( amicon ultra - 15 devices ; millipore , mass .). the combined sample was loaded onto a superose ™ 6 ( 10 × 300 mm ; ge healthcare life sciences ) equilibrated with 10 mm potassium phosphate buffer ( ph 7 . 8 ) containing 100 mm kcl . protein was eluted at a flow rate of 1 ml / min . fractions of 1 ml were collected and tested for artemisinic aldehyde double bond reductase activity ( see above ). retention times were compared with those of the following gel filtration markers ( ge healthcare life sciences ): thyroglobulin ( 669 kda ), ferritin ( 440 kda ), catalase ( 232 kda ), aldolase ( 158 kda ), bsa ( 67 kda ), ovalbumin ( 43 kda ), chymotrypsinogen ( 25 kda ), and ribonuclease a ( 14 kda ). elution was monitored at 280 nm . the molecular mass in the native state of the artemisinic alcohol double bond reductase was estimated to be 44 kda . the active fractions were combined , desalted by dialysis with 10 mm potassium phosphate containing 1 mm dtt and concentrated by ultrafiltration for subsequent purification using a batch affinity purification step as follows . reactive red 120 agarose ( type 3000 - cl , sigma ; 100 μl ) was pre - equilibrated with an equal volume of 10 mm potassium phosphate buffer ( ph 7 . 8 ) containing 2 mm zncl 2 . the buffer was removed and 500 μl of protein solution was added and gently shaken on ice for 10 min . the buffer was removed and the agarose was washed with 3 × 1 ml 10 mm potassium phosphate containing 1 mm dtt . the active protein was eluted by incubating on ice with 3 × 1 ml 10 mm potassium phosphate containing 1 mm dtt , 1 mm edta and 1 mm nadph . the eluted protein was desalted and concentrated by spin dialysis ( amicon ultra - 15 devices ; millipore , mass .). the double bond reductase preparation was subjected to sds - page ( 10 - 20 % acrylamide ) under reducing conditions followed by silver staining . the bands of interest were excised from the gel . in - gel trypsin digestion was performed according to the method of massprep digestion 5 . 0 ( waters massprep ™ station ) as follows . the gel pieces were de - stained twice with a solution containing 30 mm potassium ferricyanide , 100 mm sodium thiosulphate , 100 mm ammonium bicarbonate and 50 % ( v / v ) acetonitrile , then reduced with 10 mm dtt , followed by alkylation with 55 mm iodoacetamide . the gel pieces were destained two more times and rehydrated in digestion buffer containing 100 mm ammonium bicarbonate and 6 ng / μl trypsin ( sequencing grade , promega ). after 5 h incubation at 37 ° c ., the gel slices were extracted three times with 1 % ( v / v ) formic acid and 2 % ( v / v ) acetonitrile . the gel extracts were transferred to a 96 well pcr plate , using a massprep protein digest station ( wates / micromass , manchester , uk ) prior to lc - ms analysis . the aforementioned gel extracts containing trypsin - digested proteins were evaporated to dryness , then dissolved in 6 μl of 1 % ( w / v ) aqueous trifluoroacetic acid ( tfa ). the resulting solutions were subjected to lc - ms by injection into a nanoacquity hplc ( waters , milford , mass ., usa ) interfaced to a q - tof ultima global hybrid tandem mass spectrometer fitted with a z - spray nanoelectrospray ion source ( waters / micromass , manchester , uk ). solvent a consisted of 0 . 1 % ( v / v ) formic acid in water , while solvent b consisted of 0 . 1 % ( v / v ) formic acid in acetonitrile . the samples were first adsorbed on a c18 trapping column ( symmetry 180 μm × 20 mm ; waters ) and washed for 3 min using solvent a at a flow rate of 15 μl / min . the trapped peptides were eluted onto a c18 analytical column ( 1 . 7 μm beh130 c18 100 μm × 100 mm ; waters ). separations were performed using a linear gradient of 10 : 90 % to 45 : 55 % a : b over 45 min . the composition was then changed to 20 : 80 % a : b and held for 10 min to flush the column before re - equilibrating for 7 min at 100 % solvent a . mass calibration of the q - tof instrument was performed using a product ion spectrum of glu - fibrinopeptide b acquired over the m / z range 50 to 1900 . lc - ms / ms analysis was carried out using data dependent acquisition , during which peptide precursor ions were detected by scanning from m / z 400 to 1900 in tof ms mode . multiply charged ( 2 +, 3 +, or 4 +) ions rising above predetermined threshold intensity were automatically selected for tof ms / ms analysis , by directing these ions into the collision cell where they fragment using low energy collision - induced dissociation ( cid ) by collisions with argon and varying the collision energy by charge state recognition , product ion spectra were acquired over the m / z range 50 to 1900 . lc - ms / ms data was processed using mascot distiller ver . 2 . 1 . 1 . 0 ; matrixscience ) used to searched a local artemisia annua expressed sequence tag database ( teoh , polichuk , reed , nowak , & amp ; covello 2006 ) using mascot ( matrix science inc ., boston , mass .). searches were performed using carbamidomethylation of cysteine as a fixed modification and oxidation of methionine as a variable modification , allowing for one missed cleavage during trypsin digestion . a cdna encoding the artemisia annua artemisinic aldehyde double bond reductase designated aadbr2 , and corresponding to the est clone gstsub 026_g01 , was obtained through smart - race - pcr using gene - specific primers 5 ′- gctcataagatgcaccttaataag - 3 ′ ( seq id no . : 5 ) and adaptor pcr primer 5 ′- aagcagtggtatcaacgcagagt - 3 ′ ( seq id no . : 6 ) ( clontech laboratories , inc .) and taq dna polymerase ( invitrogen canada inc .). the resulting pcr product was cloned into pcr 2 . 1 - topo vector resulting in the plasmid the ppcr2 . 1 - aadbr2 . the dna sequence of the insert of pcr2 . 1 - aadbr2 was determined with an ab13700 dna sequencer using a bigdye terminator cycle sequencing kit ( applied biosystems ). for e . coli expression , the open reading frame ( orf ) of the aadbr2 gene was pcr - amplified using gene - specific primers 5 ′- caccatgtctgaaaaaccaaccttg - 3 ′ ( seq id no . : 7 ) and 5 ′- gctcataagatgcaccttaataag - 3 ′ ( seq id no . : 8 ), vent dna polymerase ( new england biolabs , cambridge , mass ., usa ) and the plasmid of ppcr2 . 1 - aadbr2 as the template . the resulting pcr product was cloned via the gateway entry vector pentr / d / topo ( invitrogen ) into the gateway destination vector pdest17 ( invitrogen ) to generate a bacterial expression clone pdest17 - aadbr2 . the orf of aadbr2 was in frame with vector sequence encoding an n - terminal his 6 tag . the plasmid pdest17 - aadbr2 was introduced into e . coli strain rosetta ™ 2 ( de3 ) ( novagen ) using heat shock at 42 c . transformants were grown on luria broth ( lb ) and selected on ampicillin ( 100 μg / ml ) at 37 ° c . for 24 hours . a single colony containing pdest17 - aadbr2 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 . for 4 hours with shaking . cells were centrifuged at 2 , 000 × g at 4 ° c . for 10 minutes . the resulting cell pellets 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 phenylmethylsulfonyl fluoride ( pmsf ). cells were lysed with lysozyme ( 0 . 2 mg / ml of cell suspension ) on ice for 30 minutes followed by sonication on ice with five 30 s pulses . the sonicated e . coli extract was then centrifuged at 20 , 000 × g for 15 min at 4 ° c . to obtain the supernatant as a cell - free extract containing aadbr2 . the aforementioned cell - free extract was loaded onto a his - trap ff column ( amersham bioscience , nj ) 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 volumes of binding buffer and the recombinant aadbr2 was eluted with elution buffer ( 20 mm sodium phosphate , 500 mm nacl , ph 7 . 5 ) containing increasing concentrations of imidazole in a step - wise fashion ( 50 mm , 100 mm , 200 mm , 250 mm , and 300 mm imidazole ). most of aadbr2 was eluted in the elution buffer containing 200 mm imidazole . the eluted fractions were concentrated and desalted by spin dialysis ( amicon ultra - 15 devices ; millipore , mass .) following manufacturer &# 39 ; s protocol . the purity of the recombinant aadbr2 was checked by sds - page using rapid stain ( biosciences , st . louis , mo .) for visualization . the purified recombinant his - tagged aadbr2 protein was assayed with artemisinic aldehyde , followed by gas chromatography / mass spectrometry analysis . enzyme reactions were initiated by adding the 0 . 4 mm artemisinic aldehyde to 300 μl reaction mixture containing 50 mm tris - hcl ( ph 8 . 0 ), 1 mm nadph , 2 mm dtt and 2 . 0 μg of aadbr2 . negative controls were carried out with boiled proteins , without nadph . reactions were allowed to proceed for 30 minutes at 30 ° c . with shaking , stopped by adding 15 μl acetic acid and extracted with 100 μl ethyl acetate . the ethyl acetate extracts were pooled and partially evaporated prior to gc - ms analysis . the reaction products were confirmed by comparing gc retention time and ms data with those of synthetic ( 11r )- dihydroartemisinic aldehyde . for quantification , octadecane was used as an internal standard . for comparison of the catalytic properties of aadbr2 against other substrates , reaction mixtures were pre - warmed to 30 ° c ., and reactions were initiated by addition of aadbr2 . the ph optimum of the purified aadbr2 was determined to be 7 . 5 in assay that included one of three 50 mm buffers ( mes , hepes , and tris - hcl ) adjusted to between ph 5 . 5 and 9 . 0 in 0 . 5 - unit intervals and 0 . 5 mm artemisinic aldehyde and 0 . 48 μg of purified recombinant aadbr2 . apparent kinetic parameters were determined under conditions that limited conversion to less than 10 % as follows . concentrations of artemisinic aldehyde ( 6 - 250 μm , 0 . 24 μg of aadbr2 , 2 min ), 2 - cyclohexen - 1 - one ( 80 - 10 , 000 μm , 0 . 48 μg of aadbr2 , 10 min ), and (+)- carvone ( 20 - 5000 μm , 0 . 48 μg aadbr2 , 10 min ) were varied in the presence of 1 mm nadph . to assess cofactor specificity , concentrations of nadph ( 10 - 640 μm , 0 . 48 μg of aadbr2 , 2 min ) and nadh ( 0 . 2 - 13 mm , 1 . 9 μg of aadbr2 , 2 min ) were varied in the presence of 0 . 5 mm artemisinic aldehyde . the ethyl acetate extracts from triplicate reaction mixtures were directly analyzed by gc / ms . octadecane was used as an internal standard to quantify the products formed from the reactions using response factors determined using standards for each enzyme product . k m and k cat values were determined by nonlinear regression analysis using graphpad ™ software ( graphpad ™ software inc ., san diego , calif .). as a means of testing the effect of expression of multiple genes in the artemisinin pathway in yeast , the plasmids pesc - his - fps - ads , pesc - leu - cyp - cpr and pyes - dest52 - aadbr2 were constructed as follows . all plasmids were confirmed by dna sequencing . the open reading frame of farnesyl pyrophosphate synthase ( fps ; genbank accession no . af136602 ) was isolated from a . annua plants by rt - pcr using the oligonucleotide primers 5 ′- taa gcggccgc atgagtagcatcgatctgaaatcc - 3 ′ ( seq id no . : 9 ), and 5 ′- taa actagt ctacttttgcctcttgtagattt - 3 ′ ( seq id no . : 10 ). the underlined sequences denote noti and spei restriction sites for sub - cloning , and the start and stop codons of the orf are indicated bold . the resulting pcr product was digested with noti and spei and ligated into the yeast expression vector pesc - his ( stratagene , la jolla , calif .) to give the plasmid pesc - his - fps . similarly , the open reading frame of amorpha - 4 , 11 - diene synthase ( ads ; genbank accession no . af138959 ), was amplified by rt - pcr using the oligonucleotide primers 5 ′- taa ggatcc atggaacagcaacaagaagtgatc - 3 ′ ( seq id no . : 11 ) and 5 ′- taa gggccc tctatactcataggataaacgag - 3 ′ ( seq id no . : 12 ). the bamhi - and apai - digested pcr product was ligated into the bamhi - and an apa1 - digested plasmid pesc - his - fps to give the plasmid pesc - his - fps - ads . in this construct , the ads gene was fused with a myc tag . the open reading frames of cyp71av1 ( genbank accession no . dq315671 ) and a . annua cytochrome p450 reductase ( cpr ; genbank accession no . ef104642 ) were amplified by rt - pcr . for pcr - amplification of cyp71av1 , oligonucleotide primers 5 ′- att ggatcca tgaagagtatactaaaag - caatg - 3 ′ ( seq id no . : 13 ) and 5 ′- taa gtcgac ctagaaacttggaacgagtaacaac - 3 ′ ( seq id no . : 14 ) were used ; for pcr - amplification of cpr , oligonucleotide primers 5 ′- att gcggccgc atgcaatcaacaacttccgttaag - 3 ′ ( seq id no . : 15 ) and 5 ′- tga ttaattaa ttaccatacatcacggagatatc - 3 ′ ( seq id no . : 16 ) were used . the bamhi - and sali - digested cyp71av1 pcr product was ligated into the bamhi - and an sali - digested plasmid pesc - leu ( stratagene ) to give the plasmid pesc - leu - cyp . the noti - and paci - digested cpr pcr product was ligated into the noti - and paci - digested plasmid pesc - leu - cyp to give the plasmid pesc - leu - cyp - cpr . the aadbr2 orf in pentr / d - aadbr2 was subcloned into the gateway yeast expression vector pyes - dest52 ( invitrogen ) to generate a yeast expression construct pyes - dest52 - aadbr2 through the recombination between the aforementioned pentr / d - aadbr2 and pyes - dest52 by lr reaction ( invitrogen ). the saccharomyces cerevisiae strains ( oye2 and oye3 deletion strains derived from the strain cy4 ) used in this study were provided by dr . chris m . grant ( trotter et al . 2006 ). competent cells of the oye2 and oye3 deletion strains were prepared with the s . c . easycomp ™ transformation kit ( invitrogen ) and co - transformed with pesc - his - fps - ads , pesc - leu - cyp - cpr and either pyes - dest52 ( for vector control ) or pyes - dest52 - aadbr2 ( for aadbr2 co - expression ). for additional control experiments the empty yeast expression vectors pesc - his , pesc - leu and pyes - dest52 were co - transformed into the yeast cells . yeast cultures ( 10 ml ) were grown overnight at 30 ° c . in his , leu , ura liquid dropout medium ( clontech , mountain view , calif .) containing 2 % ( w / v ) glucose . after 24 h in an orbital shaker maintained at 250 rpm , cells were collected by centrifugation and washed three times with sterile water . the cells were then resuspended to an od 600 of 0 . 8 in his , leu , ura liquid dropout medium containing 2 % ( w / v ) galactose , and grown for another 36 h . yeast cultures were then centrifuged and the medium was removed and extracted with 1 ml ethyl acetate . the yeast pellet was suspended in potassium phosphate buffer ( ph 9 . 0 ) and sonicated in a sonicating water bath for 2 min . the suspension was centrifuged . the supernatant was removed , acidified with 1 ml of 2n hcl and extracted with 1 ml ethyl acetate . the ethyl acetate fractions from the medium and yeast were pooled and 20 μg of octadecane was added as an internal standard . the ethyl acetate extracts were concentrated to dryness under a nitrogen stream and methylated by treatment with diazomethane prior to gc - ms analysis . crude enzyme extracts were initially prepared from the flower buds of a . annua line 2 / 39 and assayed for the conversion of artemisinic aldehyde to ( 11r )- dihydroartemisinic aldehyde . in this study , it was established that artemisinic aldehyde underwent reduction to afford ( 11r )- dihydroartemisinic aldehyde in the presence of nadph ( fig6 , a and b ). the enzyme responsible for the reaction , artemisinic aldehyde double bond reductase , was partially purified from the flower buds of a . annua line 2 / 39 using four steps that included fractional ammonium sulphate precipitation and ion - exchange , size exclusion and affinity chromatography . this resulted in a 1200 - fold purification on a protein basis relative to the original plant extract . the partially purified enzyme was subjected to sds - page followed by silver staining . the most densely staining bands were excised from the polyacrylamide gel , digested with trypsin , and analyzed by lc / ms . when the resulting data from a band corresponding to 43 kda was used to search a database of artemisia annua expressed sequence tags ( est ) a four peptide match was found to the est called gstsub — 50_f07 as indicated in table 1 . table 1 depicts the results of a mascot proteomics search showing five peptide signals from lc / ms / ms which match four peptide sequences predicted by the artemisia annua expressed sequence tag named gstsub — 50_f07 . to test the catalytic properties of the gene product , designated aadbr2 , corresponding to gstsub — 50_f07 , its full length cdna clone ( pdest17 - aadbr2 ) was obtained by smart - race - pcr . the nucleotide sequence of the open reading frame of the dna insert of pdest17 - aadbr2 is given in fig4 ( seq id no . 3 ). this orf encodes a 415 - amino acid protein with a predicted molecular mass of 45 . 6 kda . for initial functional studies , plasmid pdest17 - aadbr2 was introduced into the rosetta ™ 2 ( de3 ) e . coli strain ( novagen ). the transgenic rosetta ™ 2 ( de3 ) cells were grown and induced with 1 mm iptg . the recombinant aadbr2 protein was purified from cell - free extracts and the corresponding protein product including the n - terminal his tag fusion is given in fig5 ( seq id no . 4 ). the purified aadbr2 protein was assayed with various substrates followed by analysis by gas chromatography / mass spectrometry . fig6 ( c and d ) shows the results of this analysis indicating the nadph - dependent formation of ( 11r )- dihydroartemisinic aldehyde as the major product using artemisinic aldehyde as a substrate . control experiments with boiled enzyme preparation and in the absence of nadph did not support the production of dihydroartemisinic aldehyde . it is predicted that the wild type product of aadbr2 will have similar artemisinic aldehyde double bond reductase activity as the his tag fusion protein product of pdest17 - aadbr2 . aadbr2 was also tested with other potential substrates , including arteannuin b , artemisinic acid , artemisinic alcohol , artemisitene , (+)- carvone , coniferyl aldehyde , 2 - cyclohexen - 1 - one , 2e - hexenal , 2e - nonenal , (+)- α - pinene , (+)- pulegone , and sabinone . the results indicate that , in addition to artemisinic aldehyde , aadbr2 has activity on 2 - cyclohexen - 1 - one , (+)- carvone , and low activity on 2e - nonenal ( about 12 % of the rate for artemisinic aldehyde ; fig8 ). based on comparison with a standard 5 : 1 mixture of (+)- dihydrocarvone /(+)- isodihydrocarvone and assuming that the configuration at c4 was retained , the major and minor products of (+)- carvone were identified as (−)-( 1r , 4s )- isodihydrocarvone and (−)-( 1s , 4s )- dihydrocarvone , which were produced in a 5 : 1 ratio . no activity was detected with arteannuin b , artemisinic acid , artemisinic alcohol , artemisitene , coniferyl aldehyde , 2e - nonenal , (+)- α - pinene , (+)- pulegone , and sabinone . the ph optimum of aadbr2 was determined to be ph 7 . 5 . at ph 5 . 5 , the enzyme activity was completely inhibited ; at ph 9 . 0 , it retained about 25 % of its activity at ph 7 . 5 . preliminary experiments indicated that the enzyme was also active in the presence of nadh . kinetic parameters were determined for artemisinic aldehyde , 2 - cyclohexen - 1 - one , (+)- carvone , nadph , and nadh ( table 2 ). relative to 2 - cyclohexen - 1 - one and (+)- carvone , the reductase was very specific for artemisinic aldehyde , for which the k m was more than 30 - fold lower ( table 2 ). the enzyme is also highly specific for . nadph with a k m of 95 μm , as compared with more than 770 μm for nadh . in order to test the effect of expression of aadbr2 on yeast metabolism , three yeast strains were developed : a control strain containing three empty vectors ; a strain which reconstitutes the artemisinin pathway up to artemisinic acid by expressing farnesyl diphosphate synthase ( fps ), amorpha - 4 , 11 - diene synthase ( ads ) and amorpha - 4 , 11 - hydroxylase ( cyp71av1 ); and a strain which additionally expresses aadbr2 . as indicated in fig7 a , the control strain does not produce artemisinin - related compounds such as artemisinic acid or dihydroartemisinic acid . also , as expected the strain expressing cyp71av1 , but not aadbr2 , accumulated artemisinic acid , similar to a previous report ( ro et al ., 2006 ). in this case the level of artemisinic acid in was 29 . 4 (+/− 4 . 7 ) μg / ml culture . in the strain expressing aadbr2 , ( 11r )- dihydroartemisinic acid accumulated to a level of 15 . 7 (+/− 1 . 4 ) μg / ml culture ( and artemisinic acid accumulated to 11 . 8 (+/− 2 . 8 )). this demonstrates the utility of the aadbr2 gene in the production of ( 11r )- dihydroartemisinic acid . table 3 provides mutations of aadbr2 which are expected to result in proteins that retain artemisinic aldehyde double bond reductase activity . the protein may contain any one of or combination of such mutations . nucleotide substitutions which either retain the same amino acid sequence as seq id no . : 2 or changes it to one of the mutant amino acid sequences in table 3 would comprise a functional nucleic acid molecule of the present invention . such mutations may be created by methods as described in sambrook et al . 2001 and ausubel et al . eds . 2001 . altschul , s . f ., gish , w ., miller , w ., myers , e . w ., & amp ; lipman , d . j . 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( 2003 ), “ stereoselective total synthesis of (+)- artemisinin ”, tetrahedron lett , vol . 44 , pp . 387 - 389 . other advantages that are inherent to the structure are obvious to one skilled in the art . the embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed . variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims .